Incorporation of non-canonical amino acids into proteins in yeast

Fungal Genetics and Biology 89 (2016) 137–156

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Incorporation of non-canonical amino acids into proteins in yeast Birgit Wiltschi acib – Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria

a r t i c l e

i n f o

Article history: Received 16 September 2015 Revised 1 February 2016 Accepted 3 February 2016 Available online 8 February 2016 Keywords: Genetic code expansion Non-canonical amino acid Orthogonal pair Protein engineering Saccharomyces cerevisiae Pichia pastoris

a b s t r a c t Non-canonical amino acids add extraordinary chemistries to proteins when they gain access to translation. In yeast, they can be incorporated into proteins by replacing a canonical amino acid or in a site-specific manner in response to an amber stop codon. The first approach simply exploits the natural substrate tolerance of the aminoacyl-tRNA synthetases in an auxotrophic host. The latter requires the coexpression of an orthogonal aminoacyl-tRNA synthetase that is specific for the non-canonical amino acid together with an amber suppressor tRNA. This review briefly recaps the residue- and site-specific incorporation techniques for non-canonical amino acids in yeast. It describes the selection system for orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pairs and compares the different expression systems for these pairs. Numerous examples illustrate the application of non-canonical amino acids for protein engineering in yeast. The compilation includes the chemical structures of the amino acid analogs, the orthogonal pairs that were used for their incorporation and the titers of the labeled variant proteins. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Non-canonical amino acids (ncAAs) are a relatively new gem in the protein engineer’s toolbox. This is attributed to the fact that ncAAs are not prescribed by the standard genetic code such as the canonical amino acids (cAAs), but they can only participate in ribosomal translation under tightly controlled conditions. The benefits are well worth the effort, though. NcAAs comprise side chain chemistries and/or structures that are not available from the cAAs. Among them, the fluorinated amino acids (recently reviewed by Odar et al. (2015)) or those that contain reactive groups, such as carbonyl, alkene, or alkyne moieties, are of particular interest. The latter facilitate the artificial post-translational modification of proteins by bioorthogonal conjugation reactions (reviewed by

Abbreviations: aaRS, aminoacyl-tRNA synthetase; cAA, canonical amino acid; Leu5 CalB, Candida antarctica lipase B; EcLeuRS/EctRNACUA , orthogonal leucyl-tRNA Leu5 Tyr Tyr synthetase/tRNACUA pair from E. coli; EctRNA0 CUA , E. coli suppressor tRNACUA Tyr without the CCA trinucleotide at its 30 -terminus; EcTyrRS/EctRNACUA , orthogonal Tyr tyrosyl-tRNA synthetase/tRNACUA pair from E. coli; 5-FOA, 5-fluoroorotic acid; GPCR, G-protein coupled receptor; hSOD, human superoxide dismutase; MbPylRS/ Pyl Pyl MbtRNACUA , pyrrolysyl-tRNA synthetase/tRNACUA pair from Methanosarcina barkeri; Pyl Pyl MmPylRS/MmtRNACUA , orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair from Methanosarcina mazei; ncAA, non-canonical amino acid; ncAARS, non-canonical aminoacyl-tRNA synthetase; NMD, nonsense-mediated mRNA decay; o-pair, orthogonal pair; QBP, glutamine-binding protein; SAM, S-adenosylmethionine; SCS, stop codon suppression, site-specific; SPI, supplementation based incorporation, residue-specific; X-gal, 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside. E-mail address: [email protected] http://dx.doi.org/10.1016/j.fgb.2016.02.002 1087-1845/Ó 2016 Elsevier Inc. All rights reserved.

Kim et al. (2013)). Photoactivatable groups such as azides or benzophenone facilitate crosslinking studies and fluorophores can be used as environmentally sensitive probes. Simply put, the translation of ncAAs into proteins allows their purposeful and directed chemical modification. Accordingly, ncAAs have found application in in vivo structure-function studies (Cellitti et al., 2008; Schultz et al., 2006; Ye et al., 2009), to study protein–protein interaction (Berg et al., 2014; Suchanek et al., 2005; Yamano et al., 2010), protein localization (Chatterjee et al., 2013; Lee et al., 2009), to regulate protein activity (Cirino et al., 2003; Dominguez et al., 2001; Ugwumba et al., 2010; Wu et al., 2004; Zheng and Kwon, 2013) and to generate new protein function (Chin et al., 2002; Deiters and Schultz, 2005; Tippmann and Schultz, 2007; van Hest et al., 2000; Wang et al., 2003; Zhang et al., 2002). For a comprehensive collection of application examples see the reviews by Davis and Chin (2012), Dumas et al. (2015), Kim et al. (2013), Liu and Schultz (2010), Neumann (2012), Wang et al. (2009), and Zheng and Kwon (2012). The incorporation of ncAAs was reported for Escherichia coli [for recent reviews see (Dumas et al., 2015; Zheng and Kwon, 2012)], mammalian cells (Sakamoto et al., 2002; Suchanek et al., 2005) and plant cells (Li et al., 2013). As well, ncAAs were incorporated in entire animals, such as Drosophila melanogaster (Bianco et al., 2012), Caenorhabditis elegans (Greiss and Chin, 2011; Yuet et al., 2015), Bombyx mori (Teramoto and Kojima, 2014), rabbit (Gamcsik and Gerig, 1986; Westhead and Boyer, 1961), and cow (Black and Kleiber, 1955). NcAAs have also been introduced to dif-

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ferent yeasts, such as Saccharomyces cerevisiae (Bushnell et al., 2001; Chin et al., 2003a; Wiltschi et al., 2008), Pichia pastoris (Budisa et al., 2010; Young et al., 2009), and Candida albicans (Palzer et al., 2013). Yeasts combine the advantages of microbes with the capabilities of a eukaryotic organism: Genetic manipulations are relatively simple and the tools for heterologous protein expression are well established. The fermentation design is straight forward and inexpensive media allow for rapid growth to high cell densities, for instance with P. pastoris. Moreover, yeasts tolerate the overexpression of transmembrane proteins or large protein complexes, they efficiently secrete proteins into the surrounding medium, and they posttranslationally modify their proteins. These capabilities can be particularly beneficial for the expression of proteins from (higher) eukaryotic or plant sources and are largely absent in routine prokaryotic production hosts such as E. coli. Baker’s yeast S. cerevisiae and P. pastoris are the most well-known yeasts for heterologous protein production, but a number of other yeast have been established as production hosts as well (Böer et al., 2007). S. cerevisiae as well as P. pastoris enjoy GRAS status (Generally Recognized as Safe) (Böer et al., 2007; Vogl et al., 2013). These beneficial traits have spurred the expansion of the genetic code in yeasts. Currently, ncAAs can be incorporated into proteins produced in yeasts either residue- or site-specifically.

2. Residue specific incorporation of ncAAs in yeast The residue-specific incorporation of ncAAs is simple and straight forward as it exploits the substrate tolerance of the host aminoacyl-tRNA synthetases (aaRSs) and genetic manipulation of the translation apparatus is usually not necessary. The aaRS charge their cognate tRNA(s) with the appropriate cAA, nevertheless, they accept structurally and/or chemically similar amino acids. Once the tRNA is aminoacylated with the ncAA, it can participate in ribosomal translation and the ncAA is incorporated into the nascent polypeptide instead of the analogous cAA at the codons specifying it. The incorporation is a stochastic process and it is assumed that all codons encoding the same cAA have the same probability of being interpreted with the ncAA. Naturally, an amino acid analog is a much worse substrate for an aaRS than its canonical substrate (Kiick and Tirrell, 2000). Consequently, to be charged onto a tRNA the intracellular level of the ncAA must be substantially higher than that of the cAA. Strains that are auxotrophic for the cAA whose analog is to be incorporated facilitate the ‘‘remote” control of the intracellular cAA and/or ncAA levels: By supplementing the medium with a cAA or ncAA the intracellular levels of both can be controlled from outside (supplementation based incorporation, SPI). E. coli is a routine host for the residue-specific incorporation of ncAAs, for instance to manipulate the properties of enzymes (reviewed by Zheng and Kwon (2012)). Proteome-wide residuespecific incorporation of ncAAs with reactive side chains (reviewed by Johnson et al. (2010)) has been used to analyze proteomes of mammalian cells (Beatty et al., 2006; Dieterich et al., 2006; Eichelbaum et al., 2012; Landgraf et al., 2015), zebra fish larvae (Hinz et al., 2012) and targeted subsets of C. elegans cells (Yuet et al., 2015). However, reports on the residue-specific incorporation of ncAAs in yeast are rather scarce. The E. coli aaRSs show broad substrate tolerance and a number of structurally and/or chemically close analogs of the cAAs can be incorporated into (target) proteins. Although the substrate tolerance of yeast aaRSs has not been systematically analyzed, there is evidence that yeast aaRSs also accept structurally and/or chemically similar amino acid analogs. Very early, inhibitory effects of tryptophan analogs, such as L-tryptazan (1; the structures of the ncAAs mentioned in the text are shown in Table 1), on protein biosynthesis and cell growth

were observed in S. cerevisiae. The inhibition could be competitively reversed by DL-tryptophan while L-tyrosine caused only partial reversal (Halvorson et al., 1955). When grown in the presence of L-ethionine (3) this methionine analog was incorporated into proteins of S. cerevisiae and Torulopsis utilis (Candida utilis) (Maw, 1966a), and in Candida slooffii (Kazachstania slooffiae) (Mendonça and Travassos, 1972). Specific mutations conferred resistance to the detrimental effects of 3 (Maw, 1966b). These early studies indicated that in addition to 3, other Met analogs such as L-selenomethionine

(4), and most probably trifluoro-L-methionine (5) could be incorporated into yeast proteins (Colombani et al., 1975). Téllez and coworkers reported on the accelerated degradation of mitochondrial translation products from S. cerevisiae in the presence of 3 (Téllez et al., 1985). NcAAs are efficiently edited in S. cerevisiae, for instance the incorporation of the Met analog

L-homocysteine

(23) is prevented by methionyl-tRNA synthetase (MetRS) (Jakubowski, 1991). 2.1. Applications of the residue-specific incorporation of ncAAs in yeast The

residue-specific

L-selenomethionine

substitution

of

Met

residues

by

(4) has found broad application in protein crystallography. The seleno analog of Met is used for the phase determination of proteins by single-wavelength anomalous dispersion (SAD) and multi-wavelength anomalous dispersion (MAD) phasing methods (Kitajima et al., 2010). The technique is routine in E. coli and nearly 100% replacement can be achieved in methionine auxotrophic hosts (Malkowski et al., 2007). The Kornberg group labeled the RNA polymerase II of S. cerevisiae with 4 using a methionine auxotrophic yeast strain (Bushnell et al., 2001). The auxotrophic cells did not grow in the absence of Met, this is why Bushnell et al. first accumulated cell mass on medium containing only Met and then shifted the cells to medium with 4 but without Met. As RNA polymerase II was constitutively expressed this procedure resulted in 30% labeling with 4. To overcome the growth problem yet still promote labeling of the RNA polymerase with 4, the cells were grown in medium that contained Met and 4 at a molar ratio of 1:9. Otherwise, Bushnell et al. cultivated a non-auxotrophic host in the presence of 4. In either way, they were able to increase the incorporation efficiency to 65% (Bushnell et al., 2001). L-Selenomethionine (4) is toxic for yeast cells. Malkowski et al. hypothesized that the toxicity of 4 is due to the formation of the seleno derivative of S-adenosylmethionine (SAM), Seadenosylmethionine. SAM is the primary methyl-donor of the cells that is involved in important cellular processes (Thomas and Surdin-Kerjan, 1997) and either Se-adenosylmethionine per se could be toxic or one of its metabolic products (Malkowski et al., 2007). To alleviate the toxic effects of 4, Malkowski et al. blocked the biosynthesis of SAM in S. cerevisiae by deleting both SAM synthetases encoded by SAM1 and SAM2. In this way, they achieved near quantitative (>95%) replacement of Met by 4 and successfully determined the 3D structure of the tryptophanyl-tRNA synthetase of S. cerevisiae using MAD phasing techniques. However, the deletion of both SAM synthetases rendered the yeast cells auxotrophic for SAM and the expensive compound had to be supplemented in the medium (Malkowski et al., 2007). Kitajima et al. took an alternative approach to alleviate the cell toxicity of 4 (Kitajima et al., 2010). They constructed a yeast mutant that lacked high-affinity Met permease activity. As 4 is taken up into the cells by the same permease as Met, they were able to tune the intracellular levels of 4 such that efficient replacement of Met by its seleno analog occurred yet the cells were not intoxicated by 4. In this way, they achieved an incorporation efficiency of 73% of 4 in epidermal growth factor peptide, which was

Table 1 Leu5 Tyr 0 Leu5 0 0 0 Non-canonical amino acids: Structures, details on incorporation and application. [SUP4-EctRNA0 Tyr CUA]3 or [SUP4-EctRNA CUA ]3, 3 tandem copies of E. coli suppressor tRNACUA or EctRNACUA without the 3 -CCA and flanked by 5 - and 3 Tyr Leu5 0 0 Leu5 0 0 sequences of SUP4, the expression of the tandem repeats is driven by the strong PGK1 promoter. SNR52-EctRNA0 Tyr CUA-3 SUP4 or SNR52-EctRNA CUA -3 SUP4, EctRNACUA or EctRNACUA without 3 -CCA, flanked upstream by the SNR52 promoter Tyr 0 and downstream by the 30 -flanking region of SUP4. [SNR52-EctRNA0 Tyr CUA-tECUC]4, 4 tandem copies of EctRNACUA without 3 -CCA, driven by the SNR52 promoter, downstream flanked by glutamic acid tRNA (tECUC) terminator from C. albicans. In cases where an o-pair was not mentioned by name in the reference the corresponding o-pair from the literature cited in the reference is listed. BOC, bioorthogonal conjugation; feed, cells grown in presence of the ncAA; GPCR, Gprotein coupled receptor; Me ligand, novel ligand for metal ions; nnS-S, nonnatural disulfide bonds; o-pair, orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair; photoC, photo-control; PEGylation, bioorthogonal conjugation with polyethylene glycol; PIM, protein interaction mapping; PTM, posttranslational modification; SCS, stop codon suppression, site-specific; packing, effects of side-chain packing on protein folding and stability; SPI, supplementation based incorporation, residue-specific; STrep, reporter for structural transitions. Name

Structure

Analog of

Incorpo-ration method

Yeast

Translational components for incorporation

Application

References

L-Tryptazan

Trp

Feed

S. cerevisiae

Host TrpRS/tRNATrp

Biochemical effects

Halvorson et al. (1955)

2

5-Fluoro-L-tryptophan

Trp

SPI

P. pastoris

Host TrpRS/tRNATrp

Prolonged shelf-life

Budisa et al. (2010)

3

L-Ethionine

Met

Feed

T. utilis C. slooffii S. cerevisiae

Host MetRS/tRNAMet

Biochemical effects

Maw (1966b) Maw (1966a) Mendonça and Travassos (1972) Téllez et al. (1985) Colombani et al. (1975)

4

L-Selenomethionine

Met

Feed SPI

S. cerevisiae

Host MetRS/tRNAMet

Biochemical effects Protein structure elucidation Improved SPI, structure analysis Improved SPI

Colombani et al. (1975) Bushnell et al. (2001) Malkowski et al. (2007) Kitajima et al. (2010)

5

Trifluoro-L-methionine

Met

Feed

S. cerevisiae

Host MetRS/tRNAMet

Biochemical effects

Colombani et al. (1975)

6

L-Norleucine

Met

SPI

S. cerevisiae

Host MetRS/tRNAMet

SPI in S. cerevisiae

Wiltschi et al. (2008)

7

L-Homopropargylglycine

Met

SPI

S. cerevisiae

Host MetRS/tRNAMet

SPI in S. cerevisiae, BOCa

Wiltschi et al. (2008)

8

(2S)-2-amino-5(methylsulfanyl) pentanoic acid

Met

SCS

S. cerevisiae

EcLRS-DHE6/EctRNALeu5 CUA

Packinga

Brustad et al. (2008)

9

(2S)-2-amino-6-(methylsulfanyl) hexanoic acid

Met

SCS

S. cerevisiae

EcLRS-DHE6/EctRNALeu5 CUA

Packinga

Brustad et al. (2008)

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140

Table 1 (continued) Name

Structure

Analog of

Incorpo-ration method

Yeast

Translational components for incorporation

Application

References

Para-fluoro-L-phenylalanine

Phe

SPI

P. pastoris

Host PheRS/tRNAPhe

Prolonged shelf-life

Budisa et al. (2010)

11

Para-iodo-L-phenylalanine

Phe

SCS

S. cerevisiae P. pastoris

p-iodoPheRS-1/EctRNATyr CUA p-iodoPheRS-1/[SUP4-EctRNA0 Tyr CUA]3

Evolution of o-pair SCS in P. pastoris

Chin et al. (2003a) Young et al. (2009)

12

Para-azido-L-phenylalanine

Phe

SCS

S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae P. pastoris C. albicans

Evolution of o-pair BOC with alkynes Site-specific PEGylation Improved SCS SCS in P. pastoris PIM, Tsa1p and Tup1p

Chin et al. (2003a) Deiters et al. (2003) Deiters et al. (2004) Chen et al. (2007b) Young et al. (2009) Palzer et al. (2013)

13

Para-acetyl-L-phenylalanine

Phe

SCS

p-azidoPheRS-1/EctRNATyr CUA pAZ-EcRS-6b/EctRNATyr CUA Tyr EcTyrRS/EctRNACUA derivativec AzidoTyrRS-3d/[SUP4-EctRNA0 Tyr CUA]3 pAZ-EcRS-6b/[SUP4-EctRNA0 Tyr CUA]3 p-azidoPheRS-1/[SNR52-EctRNA0 Tyr CUAtECUC]4 AzidoTyrRS-3d/[SUP4-EctRNA0 Tyr CUA]3 p-acetylPheRS-1/EctRNATyr CUA p-acetylPheRS-1/[SUP4-EctRNA0 Tyr CUA]3 p-acetylPheRS-1/[SUP4-EctRNA0 Tyr CUA]3

PIM, co-chaperone Aha1 Evolution of o-pair Improved SCS BOC with alkoxyamine

Berg et al. (2014) Chin et al. (2003a) Chen et al. (2007b) Young et al. (2009)

14

Para-benzoyl-Lphenylalanine

Phe

SCS

Evolution of o-pair Improved SCS PIM, RNA polymerase II

Chin et al. (2003a) Chen et al. (2007b) Chen et al. (2007a)

PIM, GPCR SCS in P. pastoris

Huang et al. (2008) Young et al. (2009)

S. cerevisiae S. cerevisiae P. pastoris

p-benzoylPheRS-1/EctRNATyr CUA BpaTyrRS-2e/[SUP4-EctRNA0 Tyr CUA]3 p-benzoylPheRS-1/pN{GTT}PREctRNATyr CUA p-benzoylPheRS-1/EctRNATyr CUA p-benzoylPheRS-1/[SUP40 Tyr EctRNA CUA]3 p-benzoylPheRS-1/EctRNATyr CUA BpaTyrRS-2e/[SUP4-EctRNA0 Tyr CUA]3 Host TyrRS/tRNATyr

PIM, mitochondrial translocator PIM, co-chaperone Aha1 Prolonged shelf-life

Shiota et al. (2011) Berg et al. (2014) Budisa et al. (2010)

S. cerevisiae S. cerevisiae S. cerevisiae P. pastoris

S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae P. pastoris

15

Meta-fluoro-L-tyrosine

Tyr

SPI

16

O-methyl-L-tyrosine

Tyr

SCS

S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae P. pastoris

p-OMeTyrRS-1/EctRNATyr CUA OMeYRS/EctRNALeu5 CUA OMeTyrRS-1f/[SUP4-EctRNA0 Tyr CUA]3 0 OmeRSg/SNR52-EctRNA0 Tyr CUA-3 SUP4 p-OMeTyrRS-1/[SUP4-EctRNA0 Tyr CUA]3

Evolution of o-pair Evolution of o-pair Improved SCS Improved SCS SCS in P. pastoris

Chin et al. (2003a) Wu et al. (2004) Chen et al. (2007b) Wang and Wang (2008) Young et al. (2009)

17

Para-propargyloxy-Lphenylalanine

Tyr

SCS

S. cerevisiae S. cerevisiae P. pastoris

pPR-EcRS-2b/EctRNATyr CUA PR1h/[SUP4-EctRNA0 Tyr CUA]3 b pPR-EcRS-2 /[SUP4-EctRNA0 Tyr CUA]3

BOC with azides Improved SCS SCS in P. pastoris

Deiters et al. (2003) Chen et al. (2007b) Young et al. (2009)

18

(2S)-2-aminooctanoic acid

Ala

SCS

S. cerevisiae S. cerevisiae

C8RS/EctRNALeu5 CUA EcLRS-DHE6/EctRNALeu5 CUA

Evolution of o-pair Promiscuous o-pair, packinga

Wu et al. (2004) Brustad et al. (2008)

B. Wiltschi / Fungal Genetics and Biology 89 (2016) 137–156

10

Table 1 (continued) Name

Structure

Analog of

Incorpo-ration method

Yeast

Translational components for incorporation

Application

References

(2S)-2-aminononanoic acid

Ala

SCS

S. cerevisiae

EcLRS-DHE6/EctRNALeu5 CUA

Packinga

Brustad et al. (2008)

20

(2S)-2-aminodecanoic acid

Ala

SCS

S. cerevisiae

EcLRS-DHE6/EctRNALeu5 CUA

Packinga

Brustad et al. (2008)

21

(2S)-2-aminohept-6-enoic acid

Ala

SCS

S. cerevisiae

AK-1/EctRNALeu5 CUA

BOC by olefin metathesisa

Ai et al. (2010)

22

(2S)-2-aminooct-7-enoic acid

Ala

SCS

S. cerevisiae

AK-1/EctRNALeu5 CUA

BOC by olefin metathesisa

Ai et al. (2010)

23

L-Homocysteine

Cys

SCS

S. cerevisiae

EcLRS-DHE6/EctRNALeu5 CUA

nnS-S, Me liganda

Brustad et al. (2008)

24

(2S)-2-amino-5sulfanylpentanoic acid

Cys

SCS

S. cerevisiae

EcLRS-DHE6/EctRNALeu5 CUA

nnS-S, Me liganda

Brustad et al. (2008)

25

(2S)-2-amino-6sulfanylhexanoic acid

Cys

SCS

S. cerevisiae

EcLRS-DHE6/EctRNALeu5 CUA

nnS-S, Me liganda

Brustad et al. (2008)

26

L-S-(2-nitrobenzyl)

Cys

SCS

S. cerevisiae

nbCRS/EctRNALeu5 CUA

photoC, caspase activity

Wu et al. (2004)

27

L-S-ferrocenyl-cysteine

Cys

SCS

S. cerevisiae

LeuRS32/EctRNALeu5 CUA LeuRS35/EctRNALeu5 CUA

Redox-active amino acida

Tippmann and Schultz (2007)

28

L-O-crotylserine

Ser

SCS

S. cerevisiae

AK-1/EctRNALeu5 CUA

BOC by olefin metathesis

Ai et al. (2010)

cysteine

B. Wiltschi / Fungal Genetics and Biology 89 (2016) 137–156

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141 (continued on next page)

142

Table 1 (continued) Name

Structure

Analog of

Incorpo-ration method

Yeast

Translational components for incorporation

Application

References

L-O-(pent-4-en-1-yl)serine

Ser

SCS

S. cerevisiae

AK-1/EctRNALeu5 CUA

BOC by olefin metathesisa

Ai et al. (2010)

30

L-O-(4,5-dimethoxy-2nitrobenzyl)serine

Ser

SCS

S. cerevisiae P. pastoris

LeuRS BH5 T252A/EctRNALeu5 CUA LeuRS BH5 T252A/[SUP40 Leu5 EctRNA CUA ]3

photoC, protein trafficking SCS in P. pastoris

Lemke et al. (2007) Young et al. (2009)

31

(2S)-2-amino-3-({[5(dimethylamino) naphthalen-1-yl]sulfonyl} amino)propanoic acid

–

SCS

S. cerevisiae S. cerevisiae

STrep, protein unfolding Improved SCS

Summerer et al. (2006) Wang and Wang (2008)

P. pastoris

LeuRSB8T252A/[SUP4-EctRNA0 Leu5 CUA ]3 LeuRSB8T252A/SNR52-EctRNA0 Tyr CUA0 3 SUP4 0 Leu5 LeuRSB8T252A/[SUP4-EctRNA CUA ]3

SCS in P. pastoris

Young et al. (2009)

32

(2S)-3-[(6-acetylnaphthalen-1-yl)amino]-2aminopropanoic acid

–

SCS

S. cerevisiae

Anap-2C/EctRNALeu5 CUA

STrep, ligand binding

Lee et al. (2009)

33

L-Pyrrolysine

Lys

SCS

–

MmPylRS/MmtRNAPyl CUA MbPylRS/MbtRNAPyl CUA

Methanosarcina spp. Desulfitobacterium hafniense natural expansion of the genetic code

Krzycki (2005)

34

N6-[(propargyloxy) carbonyl]-L-lysine

Pyl/Lys

SCS

S. cerevisiae

Pyl MbPylRS/SctDNAArg UCU-MmtDNACUA

SCS in S. cerevisiae

Hancock et al. (2010)

35

L-N

Pyl/Lys

SCS

S. cerevisiae

Pyl AcKRSi/SctDNAArg UCU-MmtDNACUA

SCS in yeast, PTMmimica

Hancock et al. (2010)

6

-acetyllysine

B. Wiltschi / Fungal Genetics and Biology 89 (2016) 137–156

29

Table 1 (continued) Name

c d e f g h i j

Analog of

Incorpo-ration method

Yeast

Translational components for incorporation

Application

References

36

N6-trifluoroacetyl-L-lysine

Pyl/Lys

SCS

S. cerevisiae

Pyl TfaKRSj/SctDNAArg UCU-MmtDNACUA

SCS in S. cerevisiae

Hancock et al. (2010)

37

N6-{[1-(6-nitro-1,3benzodioxol-5-yl) ethoxy]carbonyl}-L-lysine

Pyl/Lys

SCS

S. cerevisiae

Pyl PCKRS/SctDNAArg UCU-MmtDNACUA

Photocaged Lysa

Hancock et al. (2010)

38

N6-{[2-(3-methyl-3Hdiaziren-3-yl)ethoxy] carbonyl}-L-lysine

Pyl/Lys

SCS

S. cerevisiae

Pyl MbPylRS/SctDNAArg UCU-MmtDNACUA

Photocrosslinkera

Hancock et al. (2010)

Effect not yet experimentally shown. pAZ-EcRS-6 and pPR-EcRS-2 are identical (Deiters et al., 2003). O-pair not specified (Deiters et al., 2004). Most probably identical to p-azidoPheRS-3 (Chin et al., 2003a) or pAZ-EcRS-3 (Deiters et al., 2003), which are identical. Most probably identical to p-benzoylPheRS-2 (Chin et al., 2003a). most probably identical to p-OMeTyrRS-1 (Chin et al., 2003a). Carries identical mutations as p-OMeTyrRS-2 (Chin et al., 2003a). Most probably identical to pPR-EcRS-1 (Chin et al., 2003a). Is identical to AcKRS-3 described by Neumann et al. (2009). Is identical to AcKRS-2 described by Neumann et al. (2008).

B. Wiltschi / Fungal Genetics and Biology 89 (2016) 137–156

a b

Structure

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144

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Table 2 Titers of variant proteins. CalB, Candida antarctica lipase B; cpVenus, circularly permutated yellow fluorescent protein; GFP, green fluorescent protein; hSOD, human superoxide dismutase; n.d., not determineda; n.s., not specified; rHSA, recombinant human serum albumin. Refer to Table 1 for additional information on the o-pairs.

a b c d e f g

Variant protein

Expression host

O-pair

Titer

% of Wildtype protein

Reference

CalB[2] CalB[10] CalB[15] cpVenus(R168[28] L178[28]) GFP(Y39[21]) GFP(Y39[22]) GFP(Y39[28]) GFP(Y39[29]) GFP(Y39[31]) hSOD[6]

P. pastoris P. pastoris P. pastoris S. cerevisiae S. cerevisiae

Host TrpRS/tRNATrp Host PheRS/tRNAPhe Host TyrRS/tRNATyr AK-1/EctRNALeu5 CUA AK-1/EctRNALeu5 CUA

64%b 32%b 49%b n.d. n.d.

Budisa et al. (2010) Budisa et al. (2010) Budisa et al. (2010) Ai et al. (2010) Ai et al. (2010)

S. cerevisiae S. cerevisiae

0 LeuRSB8T252A/SNR52-EctRNA0 Tyr CUA-3 SUP4 Host MetRS/tRNAMet

S. cerevisiae S. cerevisiae S. cerevisiae

<1 mg/L 1.5–5 mg/L 0.5 mg/L 5–10 mg/L

<8%d 13–31%d n.d. 42–63%d

Chen et Chen et Brustad Chen et

hSOD(W33[17])-His6 hSOD(W33[18])-His6 hSOD(W33[27])-His6

S. cerevisiae S. cerevisiae S. cerevisiae

<1 mg/L 0.05 mg/L 3–10 mg/L 0.3 mg/L 0.25–0.5 mg/L

<8%d 20%e 25–63%d n.d. 25–33%f

Chen et al. (2007b) Chin et al. (2003a) Chen et al. (2007b) Brustad et al. (2008) Tippmann and Schultz (2007)

hSOD(W33[30])-His6 hSOD(W33[31])-His6 hSOD(W33[32])-His6 hSOD(W33[34])-His6 hSOD(W33[35])-His6 hSOD(W33[36])-His6 rHSA(E37[11]) rHSA(E37[12]) rHSA(E37[13]) rHSA(E37[14]) rHSA(E37[16]) rHSA(E37[17]) rHSA(E37[30]) rHSA(E37[31])

S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae

Host MetRS/tRNAMet PR1/[SUP4-EctRNA0 Tyr CUA]3 AzidoTyrRS-3/[SUP4-EctRNA0 Tyr CUA]3 BpaTyrRS-2/[SUP4-EctRNA0 Tyr CUA]3 0 Tyr OMeTyrRS-1/[SUP4-EctRNA CUA]3 p-acetylPheRS-1/[SUP4-EctRNA0 Tyr CUA]3 PR1/[SUP4-EctRNA0 Tyr CUA]3 EcLRS-DHE6/EctRNALeu5 CUA AzidoTyrRS-3/[SUP4-EctRNA0 Tyr CUA]3 BpaTyrRS-2/[SUP4-EctRNA0 Tyr CUA]3 OMeTyrRS-1/[SUP4-EctRNA0 Tyr CUA]3 p-acetylPheRS-1/[SUP4-EctRNA0 Tyr CUA]3 Tyr p-acetylPheRS-1/EctRNACUA Tyr PR1/[SUP4-EctRNA0 CUA]3 Leu5 EcLRS-DHE6/EctRNACUA LeuRS32/EctRNALeu5 CUA LeuRS35/EctRNALeu5 CUA LeuRS BH5 T252A/EctRNALeu5 CUA LeuRSB8T252A/[SUP4-EctRNA0 Leu5 CUA ]3 Leu5 Anap-2C/EctRNACUA Arg Pyl MbPylRS/SctDNAUCU-MmtDNACUA Arg Pyl AcKRS/SctDNAUCU -MmtDNACUA Pyl TfaKRS/SctDNAArg UCU-MmtDNACUA p-iodoPheRS-1/[SUP4-EctRNA0 Tyr CUA]3 pAZ-EcRS-6/[SUP4-EctRNA0 Tyr CUA]3 p-acetylPheRS-1/[SUP4-EctRNA0 Tyr CUA]3 p-benzoylPheRS-1/[SUP4-EctRNA0 Tyr CUA]3 0 Tyr p-OMeTyrRS-1/[SUP4-EctRNA CUA]3 Tyr pPR-EcRS-2/[SUP4-EctRNA0 CUA ]3 LeuRS BH5 T252A/[SUP4-EctRNA0 Leu5 CUA ]3 LeuRSB8T252A/[SUP4-EctRNA0 Leu5 CUA ]3

n.d. (I) 15%c (II) 250%c 2.5%c 25–63%d 17–38%d

Wang and Wang (2008) Wiltschi et al. (2008)

hSOD[7] hSOD(N27[17])-His6 hSOD(Q16[12])-His6 hSOD(Q16[14])-His6 hSOD(Q16[16])-His6 hSOD(Q16[13])-His6 hSOD(Q16[17])-His6 hSOD(W33[9])-His6 hSOD(W33[12])-His6 hSOD(W33[14])-His6 hSOD(W33[16])-His6 hSOD(W33[13])-His6

34 mg/L 17 mg/L 26 mg/L 0.3 mg/L 3.3 mg/L 4.8 mg/L 6.3 mg/L 5.5 mg/L 15 mg/L 0.3 mg/L 5 mg/L 0.05 mg/L 3–10 mg/L 2–6 mg/L

0.8 mg/L 1.11 mg/L 1.2 mg/L 30–100 lg/L

n.d. n.d. n.d. n.d.

Lemke et al. (2007) Summerer et al. (2006) Lee et al. (2009) Hancock et al. (2010)

n.s. n.s. >150 mg/L n.s. n.s. n.s. n.s. n.s.

10%g 40–45%g 43%g >20%g >20%g >20%g 37%g 23%g

Young Young Young Young Young Young Young Young

The The The The The The The

titer titer titer titer titer titer titer

of of of of of of of

S. cerevisiae S. cerevisiae S. cerevisiae S. cerevisiae

S. cerevisiae

P. pastoris P. pastoris P. pastoris P. pastoris P. pastoris P. pastoris P. pastoris P. pastoris

Wiltschi et al. (2008) Chen et al. (2007b) Chen et al. (2007b)

al. (2007b) al. (2007b) et al. (2008) al. (2007b)

et et et et et et et et

al. al. al. al. al. al. al. al.

(2009) (2009) (2009) (2009) (2009) (2009) (2009) (2009)

the wildtype target protein was not indicated in the reference. parent CalB was 53 mg/L. the parent hSOD was 2 mg/L. The cells were supplemented with 76 mg/L Met or with 76 mg/L (I) and 760 mg/L (II) of the analog 6. the wildtype hSOD was 12–16 mg/L. the wildtype hSOD was 250 ng/mL (0.25 mg/L). the wildtype hSOD was 1.0–1.5 mg/L. the wildtype rHSA was 352 mg/L.

secreted into the growth medium. In contrast to the approach of Malkowski et al. the Met permease mutant did not require the supplementation with SAM (Kitajima et al., 2010). Wiltschi et al. demonstrated that other Met analogs than 4 can be incorporated into a target protein in a methionine-auxotrophic S. cerevisiae strain (Wiltschi et al., 2008). They incorporated L-norleucine (6) and L-homopropargylglycine (7) into their target protein, human superoxide dismutase (hSOD). In analogy to the approach described by Bushnell et al. (2001), they first grew the cells in medium with Met and in the absence of the analogs. As soon as sufficient cell mass had accumulated, the cells were transferred to medium lacking Met to starve them for this amino acid. Finally, the cells were transferred to medium containing the analogs instead of Met and protein production was induced. This procedure resulted in 5 mg/L hSOD labeled with 6 if the cells were supplemented with an excess of analog but dropped to 0.3 mg/L with standard supplementation. However, the yield of the target protein labeled with 7 was very low (0.05 mg/L; see Table 2).

Moreover, hSOD was not fully labeled, they observed up to 40% labeling with 6 and only approximately 12% of the Met of hSOD were substituted with 7. In E. coli for comparison, depending on the protein and amino acid analog, several tens of milligrams of (nearly) fully labeled target protein can be produced per liter culture (Hoesl et al., 2011). A possible explanation for the noticeably lower incorporation efficiency of ncAAs in yeast could be that the protein expression phase in yeast in the presence of the ncAA lasts much longer than in E. coli, that is roughly 24 h vs 4 h or at maximum 16 h overnight expression. As the cells are continually starved for a cAA during the expression of the target protein the turnover of cellular proteins might liberate cAAs that are preferentially incorporated (Budisa et al., 2010). Repeated feeding of the cells with the ncAA during the protein production phase could counteract this effect since the intracellular levels of the ncAA in comparison to the cAAs would be kept elevated. The group of Budisa applied the residue-specific incorporation of ncAAs to proteins produced in the methylotrophic yeast

B. Wiltschi / Fungal Genetics and Biology 89 (2016) 137–156

P. pastoris (Komagataella pastoris) (Budisa et al., 2010). This yeast is important for the industrial scale production of recombinant proteins due to tightly regulated promoters and growth to high cell densities (Cregg et al., 2009). Budisa et al. extended the shelf-life of Candida antarctica lipase B (CalB) by substituting the Trp, Phe, or Tyr residues with 5-fluoro-L-tryptophan (2), para-fluoro-Lphenylalanine (10), and meta-fluoro-L-tyrosine (15), respectively. The protein titers were substantially higher than in S. cerevisiae, CalB[2] afforded 34 mg/L and CalB[15] 26 mg/L, while the incorporation of 10 resulted in 17 mg/L CalB[10] (Table 2). For comparison, the parent CalB containing only cAA was produced at 53 mg/L. The expressed CalB contained 5 Trp, 9 Tyr, and 11 Phe residues and the incorporation of the fluorinated analogs was stochastic as it resulted in a mixture of differentially labeled proteins. Fully labeled proteins were present, however, partially labeled proteins were more prominent. The preparation of the parent protein already contained several inhomogeneous protein species that obviously originated from incompletely processed proteins during secretion. Nevertheless, the successful residue-specific incorporation of the fluorinated aromatic amino acids into CalB confirmed that the method is applicable to secreted proteins in P. pastoris. Taken together, the residue-specific incorporation of ncAAs in yeasts is still in its infancy. More incorporation studies would be necessary to tap the full potential of the technology in yeasts. Currently, the yields of ncAA-labeled proteins are quite low. In this regard, S. cerevisiae appears less suitable than P. pastoris as the latter produces protein titers in the tens of mg/L range as compared to tens of lg/L in S. cerevisiae. Possible strategies to improve the incorporation efficiency of ncAAs in yeast could be the co-expression of wildtype aaRS (Link et al., 2004; Petrovic´ et al., 2013; Tang and Tirrell, 2001) or mutant aaRS with a broadened substrate scope ((Datta et al., 2002; Kirshenbaum et al., 2002; Tang and Tirrell, 2002); reviewed in (Ngo and Tirrell, 2011)). These approaches were beneficial for the incorporation of some ncAAs in E. coli. The co-overexpression of the appropriate tRNAs might be considered as well. Feeding strategies with the ncAAs during protein expression could improve the incorporation efficiency as well as bioreactor cultures that facilitate a better control of the growth parameters than shake flask cultures.

3. Site-specific incorporation of ncAAs in yeast The residue-specific incorporation of ncAAs is an all-or-none process: All codons specifying a cAA are reassigned to an ncAA and the analog cannot be introduced at (a) selected position(s) while the cAA occupies the remaining codons. In contrast, sitespecific incorporation of ncAAs facilitates the introduction of the amino acid analog at a (single) pre-defined position in a protein. More than 100 ncAAs have been successfully incorporated sitespecifically in different organisms (reviewed by Dumas et al. (2015)). The site-specific incorporation of ncAAs requires a codon to specify it uniquely. However, the 64 codons of the genetic code, that result from all possible combinations of the four nucleic acid bases, are assigned either to a cAA or to a translation termination signal and there is no truly ‘‘blank” codon. Therefore, the meaning of a sense or a stop codon must be reassigned. The three translation stop signals (TAG, amber; TAA, ochre; TGA opal) have been the preferred codons for reassignment. They are not decoded by a tRNA but by proteinaceous release factors. To convert a stop codon into a sense codon for an ncAA, a so-called suppressor tRNA must be generated that carries the appropriate anticodon to decode one of the three stop codons (stop codon suppression, SCS). For instance, the suppressor tRNACUA can read amber (TAG) stop codons. To generate a suppressor tRNA the anticodon of a tRNA

145

for a sense codon must be mutated accordingly. tRNAs that require only single base mutations in the anticodon are preferred candidates. For instance, the mutation of the AUA anticodon, which decodes the TAT codon for Tyr, to CUA produces an amber suppressor tRNATyr CUA. The amber codon has been the preferred codon for reassignment to ncAAs (Liu and Schultz, 2010). It is the rarest stop codon in E. coli (Nakamura et al., 2000) as well as in S. cerevisiae (http:// www.kazusa.or.jp). In addition, ochre (Wan et al., 2010), opal (Zhang et al., 2004), as well as quadruplet codons (Anderson et al., 2004) were reassigned to ncAAs. The incorporation of ncAAs at sense codons (Krishnakumar and Ling, 2014; Kwon et al., 2003) has been reported but is still at an early stage. Neumann et al. developed orthogonal ribosomes in E. coli for an improved incorporation of ncAAs at amber and quadruplet codons (Neumann et al., 2010). To participate in ribosomal translation the ncAA of choice must be charged onto the suppressor tRNA by a specific aminoacyl-tRNA synthetase (see below). The aminoacylated suppressor tRNA, e.g. ncAA-tRNACUA, competes with the release factor for its cognate stop codon and this competition restricts the efficient incorporation of the ncAA. In E. coli, release factor RF1 decodes amber (TAG) and ochre (TAA) codons (Scolnick et al., 1968). The attenuation of the translation termination activity of RF1 (Wu et al., 2013) or the deletion of the coding gene in combination with amending mutations of release factor RF2, that is specific for ochre (TAA) and opal (TGA) stop codons, improved the suppression efficiency with ncAAs and facilitated the incorporation at multiple amber codons (Johnson et al., 2012, 2011). The group of George Church replaced all amber TAG codons in E. coli at 321 known instances by synonymous ochre TAA codons and deleted RF1, which also allowed the suppression of multiple amber codons with ncAAs (Isaacs et al., 2011; Lajoie et al., 2013). Similar TAG ? TAA stop codon swaps were introduced during the construction of synthetic chromosomes for S. cerevisiae (Annaluru et al., 2014; Dymond et al., 2011). This approach freed the amber codon for reassignment to ncAAs in the future, however, the deletion of the release factor to enhance amber suppression with ncAAs is barely an option in S. cerevisiae. In eukaryotes, only one release factor eRF1 decodes all three stop codons (Baierlein and Krebber, 2010) and the gene encoding yeast eRF1, SUP45, is essential for the viability of the yeast (Himmelfarb et al., 1985). Proteins site-specifically labeled with an ncAA can be easily discriminated from unlabeled variants if the protein carries a Cterminal purification tag: The tag is only expressed if the stop or quadruplet codon is read through with an ncAA (or cAA). Usually, the fidelity of the incorporation systems is high (>95%, (Wang et al., 2009)), that means preferentially the desired ncAA is incorporated in response to the stop or quadruplet codon. In contrast, no such link between the incorporation of the ncAA and the availability of a purification tag exists for proteins that were residuespecifically labeled. The homogeneity of their labeling is largely dictated by the incorporation efficiency of the ncAA. An ncAA-specific aaRSs (ncAARS) is required to aminoacylate the suppressor tRNA with the ncAA for its incorporation in response to a stop or quadruplet codon. Usually, the substrate specificity of an existing aminoacyl-tRNA synthetase is evolved such that it can recognize the ncAA (see Section 3.1). The mutant aaRS must also efficiently interact with the amber-, opal-, ochre- or four-base suppressor tRNA. However, the enzyme must not charge any of the cAAs onto the suppressor tRNA nor aminoacylate any of the host tRNAs with the ncAA to prevent its misincorporation into the host proteome. Likewise, to ensure the fidelity of the incorporation of the ncAA at a stop or quadruplet codon, the suppressor tRNA must not be a substrate for the host aaRSs. In other words, the mutant aaRS together with its ‘‘cognate”

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Fig. 1. Positive/negative selection of o-pairs in S. cerevisiae. The reporter strain MaV203 carries three Gal4p-activatable reporter constructs. GAL1::lacZ, lacZ gene from E. coli under control for the yeast pGAL1 promoter; the chromosomal integration site of the GAL1::lacZ reporter is unknown. Functional expression of lacZ results in blue coloration of the cells on medium containing X-gal. HIS3UASGAL1::HIS3@LYS2, the regulatory sequences of the HIS3 gene were replaced by the GAL1 upstream activating sequence UASG resulting in a Gal4p-regulatable pHIS3 promoter, and the reporter was integrated into the yeast genome at the LYS2 locus (Durfee et al., 1993). In the his3D200 background of strain MaV203 this should confer histidine auxotrophy. However, the HIS3UASGAL1::HIS3 reporter has sufficient residual activity even in the absence of Gal4p that the cells can grow without histidine supplementation. This can be suppressed by the addition of 3-aminotriazole (+3-AT) to histidine-free (-His) medium (Durfee et al., 1993). 3-AT is an inhibitor of the HIS3 gene product imidazole glycerol phosphate dehydratase. SPAL10UASGAL1::URA3, the pSPO13 promoter with 10 Gal4p binding sites inserted drives the expression of URA3; the SPAL10UASGAL1::URA3 reporter was integrated into the ura3-52 locus; URS1, upstream regulatory sequence (Vidal et al., 1996). Functional expression of URA3 allows cell growth on uracil dropout medium (-Ura medium) during positive selection and leads to cell death in the presence of 5-fluoroorotic acid (+5-FOA) during negative selection. The library plasmid encodes the aaRS mutant library and the suppressor tRNA. The reporter plasmid encodes the GAL4 T44am R110am mutant. Both plasmids are transformed into the reporter strain and positive selection in the presence of the ncAA as well as negative selection in the absence of the ncAA can be performed. During the positive selection, the mutant aaRS (mut aaRS) of an active o-pair charges the ncAA (or a cAA) onto the suppressor tRNA (sup tRNA) which leads to functional expression of full length Gal4p and the following read-out: Cells are viable on -Ura medium and -His +3-AT; they are inviable with 5-FOA and turn blue on X-gal. Inactive opairs will neither survive on -Ura medium nor on -His +3-AT; they will be viable on 5-FOA and white on X-gal medium. During the negative selection an active o-pair will charge a cAA onto the suppressor tRNA and the read-out will be the same as for the active o-pair during positive selection. Inactive o-pairs cannot charge a cAA onto the suppressor tRNA and show the indicated ‘‘inactive” phenotype(s). Elements for plasmid maintenance and expression in yeast: LEU2, TRP1, S. cerevisiae selection markers; 2l ori, origin of replication; pADH1, constitutive ADH1 promoter; tADH1, ADH1 terminator; tCYC1, CYC1 terminator. Elements for plasmid amplification in E. coli: pUC ori, highcopy origin of replication; Ampr, ampicillin resistance marker. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

suppressor tRNA forms a pair that is orthogonal to the aaRSs and the tRNAs of the host (Liu and Schultz, 2010). An aaRS/tRNA pair that is imported from an evolutionarily distant organism is likely to be orthogonal in the host since the tRNA identity elements evolved independently in archaea, prokaryotes, and eukarya (Giege et al., 1998). Cross-species aminoacylation is inefficient and, for instance, aaRS/tRNA pairs from archaea were shown to be orthogonal in E. coli (Wan et al., 2014; Wang et al., 2001, 2000) while those from E. coli or archaea turned out to be orthogonal in yeast or mammalian cells (Chin et al., 2003a; Mukai et al., 2008). In yeast, the Tyr tyrosyl-tRNA synthetase/tRNATyr CUA (EcTyrRS/EctRNACUA) as well Leu5 as the leucyl-tRNA synthetase/tRNACUA (EcLeuRS/EctRNALeu5 CUA ) pairs from E. coli are orthogonal. As with E. coli and mammalian cells, the pyrrolysyl-tRNA synthetase/tRNAPyl CUA pair from Methanosarcina mazei (MmPylRS/MmtRNAPyl CUA) is orthogonal in yeast (reviewed in Dumas et al. (2015)). While the pyrrolysyl-tRNA synthetase from Methanosarcina barkeri (MbPylRS) can be used in yeast, the corresponding suppressor tRNA MbtRNAPyl CUA is not orthogonal (see Section 3.2.3).

3.1. Evolution of orthogonal aaRS/suppressor tRNA pairs in yeast To equip an aaRS with specificity for an ncAA a mutant library is generated, e.g., by structure-based mutagenesis (Liu and Schultz, 2010). However, this strategy implies that the 3D structure of the aaRS is known with its bound canonical substrate. The mutant library together with its suppressor tRNA is subjected to a procedure that involves a positive round of selection for the ability of the aaRS to charge the suppressor tRNA with the ncAA and a negative selection to abolish the charging with any cAA (Liu and Schultz, 1999). In case the tRNA of the imported orthogonal pair (o-pair) does not carry an anticodon for the stop or quadruplet codon, its anticodon must be mutated and the orthogonality of the pair can be assessed using the same positive and negative selection strategy as outlined above for the mutant aaRS (Wang and Schultz, 2001). Positive/negative selection procedures were described for E. coli and S. cerevisiae (reviewed in Liu and Schultz (2010) and Wang and Schultz (2004)). In E. coli, the positive selection of the aaRS library usually involves the suppression of an amber codon at a nonessential

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Table 3 EcTyrRS mutants for the site-specific incorporation of different Phe and Tyr analogs (see Table 1). Numbers in bold designate the positions in the amino acid binding pocket that were subjected to random mutagenesis; aaRS, aminoacyl-tRNA synthetase.

a

Fig. 2. Expression constructs for o-pairs. (A) The first generation expression system described by Chin et al. (2003a) carried a single tRNACUA gene under a cryptic promoter. The ncAARS was expressed from a strong constitutive ADH1 promoter (pADH1). (B) Chen et al. (2007b) improved the expression of the o-pair, primarily of the tRNACUA. These authors used tRNACUA without its native 30 -CCA and fused it to the 50 - and 30 -flanking regions of the yeast pol III gene SUP4 (50 -SUP4, 30 -SUP4). Three tandem copies of the tRNA-chimera were transcribed under the control of a strong constitutive PGK1 promoter (pPGK1). (C) Wang and Wang (2008) took a different approach to improve the expression of the suppressor tRNACUA. They fused the SNR52 promoter (pSNR52) with upstream A- (A) and B-boxes (B) to the 30 -CCAless tRNACUA. Only a single copy of the tRNA-chimera was sufficient for efficient amber suppression. (D) Young et al. (2009) adapted the construct of Chen et al. (2007b) for P. pastoris. They used the methanol-inducible FLD1 promoter (pFLD1) from P. pastoris to drive the expression of the ncAARS and optimized the Kozak (Koz) sequence. They also attached a C-terminal hexahistidine-tag (H) to facilitate the expression analysis. Finally, the backbone vector was adapted for integration into the P. pastoris genome. They did not modify the tandem expression cassette for the suppressor tRNACUA. Yeast elements: ARG4 and TRP1 are selection markers for P. pastoris and S. cerevisiae; pGPD, strong constitutive GPD (TDH3) promoter; tADH1, ADH1 terminator; tAOX1, AOX1 terminator; tCYC1, CYC1 terminator. Elements for plasmid amplification in E. coli: pUC ori and ColE1 are high-copy origins of replication; Ampr, ampicillin resistance marker; Kanr, kanamycin resistance marker.

position (permissive site) of an antibiotics resistance marker in the presence of the ncAA. The suppression of the amber codon by the incorporation of the ncAA or a cAA results in resistance toward the appropriate antibiotic. The mutant aaRSs that aminoacylate cAAs are sorted out in the following negative selection round. Only those mutant aaRSs survive this selection that do not charge the suppressor tRNA with a cAA and therefore avoid suppression of an amber codon at a permissive site in a cytotoxic protein ((Wang, 2002) as cited in (Wang and Schultz, 2004)). The use of a fluorescence marker such as GFP facilitates the selection, e.g. by fluorescence-based cell sorting (FACS) (Santoro et al., 2002).

aaRS Residue

37

EcTyrRS

Tyr Asn Asp Phe Leu Tyr

126 182 183 186 Substrate Reference

p-acetylPheRS-1a p-azidoPheRS-1 pAZ-EcRS-6 pPR-EcRS-2 p-benzoylPheRS-1 p-OMeTyrRS-1 p-iodoPheRS-1

Ile Leu Thr Thr Gly Val Val

Asn Asn Asn Asn Asn Asn Asn

Gly Ser Ser Ser Gly Ser Ser

Met Met Ala Ala Phe Met Tyr

Ala Ala Leu Leu Ala Leu Leu

13 12 12, 17 17, 12 14 16 11

UniProtKB P0AGJ9 (SYY_ECOLI) Chin et al. (2003a) Chin et al. (2003a) Deiters et al. (2003) Deiters et al. (2003) Chin et al. (2003a) Chin et al. (2003a) Chin et al. (2003a)

Contains also an Asp165Gly mutation.

Recently, Kuhn et al. described a fluorescence-based selection system where a GFP amber mutant was employed for negative and positive selection using FACS (Kuhn et al., 2010). Usually, several rounds of positive and negative selection are performed to select the mutant aaRS with the desired specificity. A similar positive/negative selection regime was developed for S. cerevisiae (Chin et al., 2003b) (Fig. 1). It makes use of the yeast transcription activator Gal4p to drive the expression of appropriate reporter genes for positive and negative selection. The codons for threonine and arginine at positions 44 and 110 of Gal4p are replaced by amber codons. These amber mutations occupy nonessential positions in full length GAL4, that is, incorporation of any amino acid at this site would not impair the function of the transcription activator (Chin et al., 2003b). Moreover, the residues which are critical for transcriptional activation lie downstream of the amber mutations so that truncated products produced in the absence of suppression would not activate the reporter genes (Brent and Ptashne, 1985; Chin et al., 2003b; Johnston and Dover, 1988; Keegan et al., 1986; Ma and Ptashne, 1987). GAL4 T44am R110am is expressed from a multi-copy 2l plasmid under the control of a strong ADH1 promoter (Fig. 1). This ensures that the transcription activator is highly active and it extends the dynamic range of the reporters (Chin et al., 2003b). The positive/negative selection strategy in yeast links the activity of an orthogonal aaRS/suppressor tRNACUA pair with the suppression of both amber sites of GAL4 T44am R110am. In case the suppression is successful, functional Gal4p drives the expression of three Gal4p-activated reporter genes in yeast strain MaV203 (Fig. 1). This reporter strain carries a chromosomal copy of a Gal4p-activatable allele each of lacZ (GAL1::lacZ), HIS3 (HIS3UASGAL1::HIS3@LYS2 in a his3D200 background) and URA3 (SPAL10UASGAL1::URA3) and it is deleted for the cellular GAL4 and GAL80 genes (Chin et al., 2003b). MaV203 is co-transformed with the o-pair library and the GAL4 T44am R110am reporter plasmid. All three reporters can be used for positive selection of o-pairs in the presence of an ncAA. The suppression of T44am and R110am of GAL4 with either an ncAA or a cAA leads to the expression of functional Gal4p, which activates the reporter genes (Fig. 1). Expression of lacZ in the presence of X-gal (5-bromo-4-chloro-3indolyl-b-D-galactopyranoside) facilitates the colorimetric detection of blue colonies, and cells expressing HIS3 and URA3 can grow on medium lacking histidine or uracil. Moreover, the activity of the gene product of HIS3 can be modulated in a dose-dependent manner by addition of 3-aminotriazole (3-AT) to the medium. Cells carrying inactive o-pairs will not survive this selection round due to the lack of functional HIS3 and URA3 and they will be white as lacZ is not expressed. To rid the library of mutant aaRSs that charge cAAs onto the suppressor tRNA, the negative selection is performed in the absence of the ncAA in medium containing 0.1% 5-fluoroorotic acid (5-FOA). The incorporation of a cAA into GAL4

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T44am R110am again will lead to the expression of functional Gal4p, however, the expression of the URA3 gene in the presence of 5-FOA is toxic and kills the cells carrying non-specific o-pairs (Fig. 1). As described for E. coli above, the positive/negative selection is repeated for several rounds to enrich ncAA-specific opairs. A detailed protocol for the selection of orthogonal pairs in yeast can be found in Cropp et al. (2007). With the described positive/negative selection strategies, large o-pair libraries can be screened in E. coli or S. cerevisiae. The ncAA-specific o-pairs can then be transferred to other organisms, such as Mycobacterium tuberculosis (from E. coli) (Wang et al., 2010), mammalian cells (from yeast) (Liu et al., 2007), or animals (Drosophila; from E. coli) (Bianco et al., 2012). 3.2. Applications of the site-specific incorporation of ncAAs in yeast A wealth of applications of site-specific incorporation of ncAAs has been reported for E. coli and the technique has received much attention for biological studies in mammalian cells and animals (reviewed in Davis and Chin (2012), Dumas et al. (2015), Neumann (2012) and Wang et al. (2009)). In this section, I will review the applications in different yeast species. 3.2.1. O-pairs for S. cerevisiae that are derived from EcTyrRS/ EctRNATyr CUA Chin et al. first expanded the genetic code of S. cerevisiae (Chin et al., 2003a). They demonstrated the utility of their positive/negative selection strategy with the EcTyrRS/EctRNATyr CUA pair ((Chin et al., 2003b); for details see the previous section). To change the substrate specificity of the EcTyrRS they chose amino acid residues that were in close proximity to the bound Tyr in the binding pocket. They randomized residues Tyr37, Asn126, Asp182, Phe183, and Leu186 of EcTyrRS and expressed the mutant library under the control of a strong ADH1 promoter from a high-copy 2l plasmid that also carried the EctRNATyr CUA (Chin et al., 2003a) (Fig. 2A). Applying their positive/negative selection strategy, they evolved o-pairs (listed in Table 1) that were specific for paraiodo-L-phenylalanine (11; p-iodoPheRS-1/EctRNATyr CUA), which contains a heavy atom in the side chain; for the reactive Phe analogs para-azido-L-phenylalanine (12; p-azidoPheRS-1/EctRNATyr CUA) and para-acetyl-L-phenylalanine (13; p-acetylPheRS-1/EctRNATyr CUA); for the photocrosslinker para-benzoyl-L-phenylalanine (14; p-benzoylPheRS-1/EctRNATyr CUA); and for the structure/function probe O-methyl-L-tyrosine (16; p-OMeTyrRS-1/EctRNATyr CUA). Table 3 provides an overview of the mutations that were introduced into EcTyrRS to generate the specificity for the different ncAAs. Chin and coworkers validated the function of their evolved o-pairs with the model protein human superoxide dismutase. They obtained 50 ng/mL of purified hSOD labeled with 13 (Chin et al., 2003a) (Table 2). Chin et al. used a mutant in which the Trp codon at position 33 was mutated to an amber codon and for the facilitated purification the protein carried a C-terminal hexahistidine-tag (hSOD(W33am)-His6). Since its first application for this purpose, this model protein has been extensively used to assess the functionality of various o-pairs in yeast (Ai et al., 2010; Brustad et al., 2008; Chen et al., 2007b; Chin et al., 2003a; Deiters et al., 2003; Hancock et al., 2010; Lee et al., 2009; Lemke et al., 2007; Nehring et al., 2012; Summerer et al., 2006; Tippmann and Schultz, 2007; Wu et al., 2004). The hSOD(Q16am)-His6 and hSOD(N27am)-His6 mutants were employed to study the efficiency of amber suppression at different positions in the protein (Chen et al., 2007b). Deiters et al. used the approach described by Chin et al. (2003a) to generate an o-pair for the chemically reactive amino acid para-propargyloxy-L-phenylalanine (17) (Deiters et al., 2003). They randomized the same residues of EcTyrRS (see above) and applied the positive/negative selection developed by Chin et al. (2003a).

They screened their mutant library not only against 17 but also against para-azido-L-phenylalanine (12). However, new mutant aaRSs for 12 did not emerge from the selection and the isolates carried the same mutations as the analogous enzymes of Chin et al. (2003a) (compare Table 1 in the Supporting Information of Deiters et al. (2003) with the supplementary Table S1 of Chin et al. (2003a)). The individual screenings with 12 and 17 yielded mutant aaRSs that showed relaxed substrate specificity for both ncAAs. An identical mutant emerged from both selections and was used for the incorporation of 12 and 17 into hSOD(W33am)His6 (pAZ-EcRS-6 aka pPR-EcRS-2, see the o-pairs with EctRNATyr CUA in Table 1; see Table 3 for mutations). Deiters et al. demonstrated the bioorthogonal conjugation of the reactive side-chains of 12 and 17 with alkyne- and azido-functionalized fluorophores, respectively, by copper(I)-catalyzed azide-alkyne cycloaddition (‘‘click chemistry” (Meldal and Tornøe, 2008)). In the same way, they functionalized hSOD(W33a[12])-His6 with polyethylene glycol (Deiters et al., 2004). In the initial system (Fig. 2A), Chin et al. expressed the EcTyrRS/ EctRNATyr CUA pair and the o-pairs that were derived from it from a high-copy 2l plasmid. While the EcTyrRS was expressed under control of the ADH1 promoter (Chin et al., 2003b), the expression of the heterologous E. coli tRNATyr CUA was most likely driven by a cryptic promoter on the plasmid or in the gene (Chen et al., 2007b). However, the transcription of tRNAs and their processing is entirely different in pro- and eukaryotes (Nakanishi and Nureki, 2005). In E. coli, tRNAs are transcribed by the sole RNA polymerase through promoters upstream of the tRNA gene (Travers, 1984). In eukaryotes like yeast, the expression of the tRNAs is driven by RNA polymerase III (pol III) (Travers, 1976). Pol III initiates transcription at a split promoter consisting of an A- and a B-box that are internal to the tRNA structural gene ((Galli et al., 1981); for a comprehensive overview of pol III – dependent genes in ten different yeast species see (Marck et al., 2006)). EctRNATyr CUA contains a functional B-box but only six residues of the A-box match the eukaryotic consensus A-box (Edwards and Schimmel, 1990). Bacterial tRNAs that diverge from the split promoter are neither efficiently transcribed nor correctly processed in eukaryotic cells and must be adapted (Sakamoto et al., 2002). However, since the A- and B-boxes are internal to the tRNA genes a modification can cripple the tRNA gene. The posttranscriptional processing of eukaryotic and prokaryotic tRNAs differs as well: The 30 -CCA repeat is added enzymatically in yeasts, while in E. coli it is encoded on the tRNA gene (reviewed in Deutscher (1984)). Chen et al. devised a strategy for the improved expression of bacterial tRNAs in yeast that takes these differences into account (Chen et al., 2007b) (Fig. 2B). They fused the heterologous bacterial tRNA to pol III-regulated up- and downstream sequences. They chose the S. cerevisiae SUP4 gene (tY(GUA)J2) which encodes the tyrosine tRNA SctRNATyr GUA and has a well characterized pol III promoter (Allison et al., 1983). As the 30 -CCA is added enzymatically in yeast, the EctRNATyr CUA without the CCA trinucleotide at its 0 30 -terminus (EctRNA0 Tyr CUA) was fused to the 55 bp upstream (5 ) 0 and 30 bp downstream (3 ) flanking sequences of the SUP4 gene (Chen et al., 2007b) (Fig. 2B). Up to six tandem copies of this SUP4-EctRNA0 Tyr CUA chimera were cloned downstream of the strong RNA polymerase II PGK1 promoter to further enhance the expression of the heterologous tRNA. Three tandem copies of the chimera under the PGK1 promoter ([SUP4-EctRNA0 Tyr CUA]3) turned out to be optimal for amber suppression while six copies slowed yeast growth and lowered the titer of the labeled target protein. To demonstrate the general applicability of their improved expression approach, Chen and coworkers generated new o-pairs (see Table 1) by combining [SUP4-EctRNA0 Tyr CUA]3 with the existing mutant aaRSs for para-azido-L-phenylalanine (12) (Chin et al., 2003a; Deiters et al., 2003), para-propargyloxy-L-phenylalanine (17) (Deiters

B. Wiltschi / Fungal Genetics and Biology 89 (2016) 137–156

et al., 2003), as well as for para-acetyl-L-phenylalanine (13), parabenzoyl-L-phenylalanine (14), and O-methyl-L-tyrosine (16) (Chin et al., 2003a). With these improved o-pairs they obtained 1–10 mg/L of hSOD(W33am)-His6 labeled with the different analogs (Chen et al., 2007b) (Table 2). The group of Wang further improved the expression of o-pairs (Wang and Wang, 2008) (Fig. 2C). They also worked with the 0 EctRNATyr CUA that lacked the 3 -CCA. However, they chose the pol III promoter of yeast SNR52 gene to drive the transcription of EctRNA0 Tyr CUA. The SNR52 promoter contains the A- and B-boxes upstream of the coding region. They are an integral part of the primary transcript but they are removed during posttranscriptional processing. Wang et al. constructed a chimeric tRNA expression cassette in which the EctRNA0 Tyr CUA was flanked by an upstream SNR52 promoter and the 30 -flanking sequence of the SUP4 gene (SNR520 EctRNA0 Tyr CUA-3 SUP4). They placed the tRNA expression cassette onto a 2l plasmid together with EcTyrRS whose expression was driven by a strong GPD promoter (Fig. 2C). The resulting orthogonal 0 EcTyrRS/SNR52-EctRNA0 Tyr CUA-3 SUP4 pair was able to suppress an amber codon at position 39 of GFP with Tyr much more efficiently (Wang and Wang, 2008) than when the SUP4 50 -flanking region drove the transcription of EctRNA0 Tyr CUA as described by Chen et al. (2007b). Although the SUP4 50 -flanking region provoked higher transcription levels of EctRNA0 Tyr CUA than the SNR52 promoter the processing and/or modification of EctRNA0 Tyr CUA expressed from the this promoter was much more efficient (Wang and Wang, 2008). Wang et al. also tackled the question whether the amber suppression efficiency could be further improved in a yeast strain that was deficient for nonsense-mediated mRNA decay (NMD) (Wang and Wang, 2008). In yeast, NMD mediates the accelerated decay of mRNAs that contain premature stop codons (Roy and Jacobson, 2013). If the NMD could be inactivated, the life-time of the target mRNA containing the amber stop codon could be prolonged which would most probably lead to an improved amber suppression. Indeed, amber suppression was improved in an upf1D knockout strain, which was deficient for NMD (Wang and Wang, 2008). However, the beneficial effect of NMD deletion is positionally biased as it is more pronounced the closer the in-frame amber codon is located to the N-terminus (Wang et al., 2009). By their improved system, Wang et al. obtained tens of milligrams of ncAA-labeled protein per liter in yeast (Wang and Wang, 2008) (Table 2, GFP(Y39[31])). The photocrosslinker amino acids para-azido-L-phenylalanine (12) and para-benzoyl-L-phenylalanine (14) have been extensively employed for protein interaction studies in yeast. Photocrosslinking with 14 was successfully used to study a G-protein coupled receptor (GPCR) in its native membrane environment (Huang et al., 2008). Huang et al. used the p-benzoylPheRS-1/EctRNATyr CUA pair described by Chin et al. (2003a) to incorporate 14 into eight specific positions of the yeast GPCR Ste2p. Ste2p is located on the cell surface where it senses the pheromone alpha factor (Naider and Becker, 2004). The amber mutations were introduced into the loop regions as well as the transmembrane domains of Ste2p. The readthrough efficiency of the amber mutations was context dependent since five of the eight amber mutants were expressed as full length Ste2p in the absence of 14. The function of two amber mutants was impaired by the incorporation of 14. In general, the expression of the mutant GPCRs was low and amounted only to about 5–10% of the wildtype receptor (Huang et al., 2008). Obviously, the free amino acid 14 was only inefficiently taken up by the yeast cells. The use of a Met-14 dipeptide clearly enhanced the uptake of the ncAA and its incorporation into the GPCR amber mutants. The beneficial effect of the dipeptide on uptake was particularly pronounced at low concentrations (0.1 mM). At higher concentrations (0.5 or 2 mM), the incorporation efficiency was comparable between the dipeptide and the free ncAA. The suppression of the

149

amber codons was not 100% efficient and truncated versions of the GPCR were produced. However, they did not adversely interfere with the full-length receptor. Huang et al. used the 14-labeled GPCR to photocapture a biotinylated alpha factor. The Hahn group used 14 for crosslinking studies with the translation machinery (Chen et al., 2007a; Mohibullah and Hahn, 2008). They incorporated 14 at surface positions of RNA polymerase II and mapped the location of the interactions with the transcription factors TFIIB, TFIIF, and TFIIE (Chen et al., 2007a). They also incorporated 14 into surface positions of the TATA-binding protein (TBP) and mapped the physical interactions of TBP with transcriptional co-regulator subunits and with the general transcription factor TFIIA (Mohibullah and Hahn, 2008). In this way, they were able to demonstrate the direct interaction between TBP and the coactivator subunits Spt3 and Spt8 of the SAGA complex (Mohibullah and Hahn, 2008). They initially used the 14-specific o-pair developed by Chin et al. (2003a), however, they found that the suppression efficiency was low because the expression of the EctRNATyr CUA was poor (Chen et al., 2007a). Similar to the reports by Chen et al. (2007b) and Wang and Wang (2008), they tested several upstream flanking sequences of S. cerevisiae tRNA genes to improve the transcription of the suppressor tRNA. They found that the medium upstream enhancing motif of the N{GTT}PR tRNA gene (which encodes tRNAAsn improved EctRNATyr expression AAC) CUA and amber suppression efficiency. The new orthogonal p-benzoylPheRS-1/pN{GTT}PR-EctRNATyr CUA pair facilitated nearly normal expression levels of some of their amber mutants with 14 (Chen et al., 2007a). Berg et al. used the improved o-pairs from Chen et al. (2007b) for the incorporation of two different photocrosslinking ncAAs into the same protein. They used the p-azidoPheRS-3/[SUP4EctRNA0 Tyr CUA]3 pair (Chen et al., 2007b) to incorporate paraazido-L-phenylalanine (12) at eight different positions in the co-chaperone Aha1 in S. cerevisiae (Berg et al., 2014). 12 was well incorporated at all positions of Aha1, though some background incorporation of cAAs occurred in the absence of 12. At selected positions, they also incorporated 14 using the p-benzoylPheRS-2/ [SUP4-EctRNA0 Tyr CUA]3 pair (Chen et al., 2007b). They found that the crosslinked product was independent of the type of photoreactive amino acid. However, they were unable to identify an interaction of Aha1 with Hsp90 in the crosslinking experiments, which had been described before. Rather, Aha1 formed homodimers (Berg et al., 2014). In contrast to Berg et al. (2014), Nehring et al. were unable to incorporate 12 into hSOD(W33am)-His6 despite the fact that they used the same expression construct for the o-pair. They expressed the corresponding mutant EcTyrRS (Chen et al., 2007b) and two other ncAARSs with reported specificity for 12 (Chin et al., 2003a; Deiters et al., 2003) in E. coli and analyzed the affinity of the isolated enzymes for 12 in comparison to Tyr. They found that all enzymes recognized Tyr under the given assay conditions but not 12 (Nehring et al., 2012). Although the contradicting observations could not be conclusively explained they emphasize the importance of the application and evaluation of o-pairs in different research environments. Several studies report the successful application of 14 to analyze protein–protein interactions in the mitochondrial protein import machinery. Shiota et al. probed different positions in the three main domains of Tom22, a multifunctional subunit of the mitochondrial protein import complex TOM40 (Shiota et al., 2011). They used the 14-specific p-benzoylPheRS-1/EctRNATyr CUA pair described by Chin et al. (2003a) and observed roughly a 50:50 ratio of full length and truncated Tom22 upon amber suppression with 14. Nevertheless, the approach was suitable to analyze the interaction of Tom22 with the other subunits of the TOM40 complex and to provide ‘‘interaction-map snapshots for membrane–protein

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Table 4 EcLeuRS mutants for the site-specific incorporation of different ncAAs. Numbers in bold designate the positions in the amino acid binding pocket that were subjected to random mutagenesis. Residues in bolditalic were not included in the mutagenesis; aaRS, aminoacyl-tRNA synthetase.

a

aaRS Residue

40

41

252

499

527

537

Substrate

Reference

EcLeuRS nbCRS LeuRS BH5 T252A OMeYRS C8RS EcLRS-DHE6 AK-1a LeuRSB8T252A LeuRS32 LeuRS35

Met Trp Gly Leu Val Ile Val Ala Gly Gly

Leu Ser Gln Glu Met Leu Ser Asn Lys Glu

Thr Thr Ala Thr Thr Thr Arg Ala Thr Thr

Tyr Ile Leu Arg Leu Ile Ser Ile Ser Arg

Tyr Ala Gly Ala Leu Ala Leu Gly Ala Ile

His Gly Phe Gly Gly Gly Gly Thr Gly Gly

Leu 26 30 16 18 8, 9, 23, 24, 25, 18, 19, 20 21, 22, 28, 29 31 27 27

UniProtKB P07813 (SYL_ECOLI) Wu et al. (2004) Lemke et al. (2007) Wu et al. (2004) Wu et al. (2004) Brustad et al. (2008) Ai et al. (2010) Summerer et al. (2006) Tippmann and Schultz (2007) Tippmann and Schultz (2007)

Contains a Glu20Lys mutation that was not included in the original library.

complexes at work” (Shiota et al., 2011). Tamura et al. (2009) and Yamano et al. (2010) followed a similar strategy to map protein–protein interactions of the mitochondrial protein import machinery. It is not clear which o-pair Tamura et al. used. They indicate the use of ‘‘p6xtRNA” described by Chin et al. (2003a), which implies six copies of suppressor tRNA. However, (Chin et al., 2003a) reported the p-benzoylPheRS-1/EctRNATyr CUA pair for the incorporation of 14 which employs only a single copy of EctRNATyr CUA. A detailed protocol for cellular interaction analyses by photocrosslinking using 14 is available from Shiota et al. (2013).

3.2.2. O-pairs for S. cerevisiae that are derived from EcLeuRS/ EctRNALeu5 CUA Next to the orthogonal EcTyrRS/EctRNATyr CUA pair the EcLeuRS/ EctRNALeu5 CUA pair from E. coli has been used to evolve a number of o-pairs in yeast. Wu et al. first explored this alternative o-pair for S. cerevisiae. They used the amber suppressor tRNALeu5 CUA from E. coli, which was derived from tRNALeu UUA ((Yamaizumi et al., 1980); GenBank accession K00225) with upstream and downstream flanking sequences. However, the source of the flanking regions was not indicated (Wu et al., 2004) (see ‘‘Library construction and selection” in the Supporting Information). The upstream flanking region aligns with pre-tRNA-Tyr from E. coli (GenBank accession X70134) and the downstream sequence appears to be from E. coli as well (B. W., personal observation). EctRNALeu5 CUA has A- and B-boxes that match the eukaryotic consensus sequences (Wang and Wang, 2008). tRNALeu5 CUA with its flanking regions and EcLeuRS were expressed from an analogous vector construct as described for the EcTyrRS/EctRNATyr CUA pair by Chin et al. (2003b). In analogy to the work of Chin et al. (2003a), Wu and coworkers chose the five amino acid residues Met40, Leu41, Tyr499, Tyr527, and His537 in the binding pocket of EcLeuRS and randomized them. Then they subjected their EcLeuRS mutant library to the positive/negative selection strategy described by Chin et al. (2003b). In this way, they evolved the o-pair nbCRS/EctRNALeu5 CUA (for relevant mutations refer to Table 4) for the site-specific incorporation of L-S-(2-nitrobenzyl)cysteine (26) (Wu et al., 2004). 26 is a photocaged Cys analog where the thiol group is protected by the photolabile ortho-nitrobenzyl group. UV irradiation cleaves the benzylic C-S bond and the free thiol group is revealed (Philipson et al., 2001; Wu et al., 2004). Wu et al. used 26 to photo-control the activity of the human proapoptotic protein caspase 3. They substituted the active site residue Cys163 with 26, which abolished the activity of the enzyme. Photolysis of 26 at position 163 by irradiation with UV light deprotected the Cys and restored the caspase activity (Wu et al., 2004). Irradiation with short wave UV light (<365 nm) restricts the application of photocaged amino acid analogs in living cells due to the cell-damaging effects. Lemke et al. sought to evolve an o-pair for the site-specific incorporation of the photocaged serine

derivative L-O-(4,5-dimethoxy-2-nitrobenzyl)serine (30), which can be activated by long wave UV light (Lemke et al., 2007). The 4,5-dimethoxy-2-nitrobenzyl group has a high quantum yield for photocleavage and its absorption spectrum is strongly redshifted. It can be photoactivated with visible blue light such as the blue laser light of a standard laser-scanning microscope (Lemke et al., 2007). Lemke et al. used the EcLeuRS/EctRNALeu5 CUA pair developed by Wu et al. (2004) and randomized the same residues of the EcLeuRS. They used the positive/negative selection devised by Chin et al. (2003b) to screen their library against 30. However, the o-pair that emerged from the screen showed high background incorporation of Leu and Ile in the absence of 30. To improve the fidelity of their o-pair, Lemke and coworkers introduced the additional Thr252Ala mutation in the editing domain of EcLeuRS. Indeed, this additional mutation resulted in the high-fidelity orthogonal LeuRS BH5 T252A/EctRNALeu5 CUA pair which was specific for 30 (see Table 4 for the mutations in LeuRS BH5 T252A and Table 2 for protein titer of hSOD(W33[30])-His6). Lemke et al. applied 30 to photo-control the subcellular localization of the yeast transcription factor Pho4 in live yeast cells. The subcellular localization of Pho4p in the nucleus or cytosol of S. cerevisiae is dependent on the intracellular inorganic phosphate levels. The phosphorylation of specific serine residues of Pho4p triggers the subcellular trafficking of the protein between these two compartments. 30 was incorporated in response to different amber mutations that had been introduced at the positions of those serine residues that were involved in phosphorylation and Pho4p translocation. The photocaged serine at these positions prevented translocation, yet blue-light induced photolysis deprotected it so that phosphorylation could occur and trigger translocation. The sitespecific incorporation of 30 and its deprotection with visible light allowed to monitor the kinetics of protein trafficking in real time (Lemke et al., 2007). 30 might be used for other applications where serine is involved in a specific binding interaction or catalytic activity. In addition to the first o-pair for L-S-(2-nitrobenzyl)cysteine (26; see above) in yeast, Wu et al. evolved the EcLeuRS/EctRNALeu5 CUA pair for the site-specific incorporation of O-methyl-L-tyrosine (16) and of (2S)-2-aminooctanoic acid (18) (Wu et al., 2004) (OMeYRS/ Leu5 EctRNALeu5 CUA and C8RS/EctRNACUA , the ncAARSs are listed with their mutations in Table 4). Starting from the EcLeuRS/EctRNALeu5 CUA pair of Wu et al. (2004), Brustad and coworkers developed an o-pair that showed polysubstrate specificity for several structurally related ncAAs. The EcLRS-DHE6/EctRNALeu5 CUA pair (mutations are shown in Table 4) was able to promiscuously insert several long chain analogs of Ala (18, 19, 20), Met (8, 9), and Cys (23, 24, 25) at the amber codon of the model protein hSOD(W33am)-His6 (Brustad et al., 2008) (refer to Table 2 for titers of the hSOD(W33[9])-His6 and hSOD(W33[18])-His6 variant proteins). The long-chain Cys analogs could form nonnatural disulfide bonds or serve as novel ligands for metal ions. NcAAs with long aliphatic site chains could be applied

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B. Wiltschi / Fungal Genetics and Biology 89 (2016) 137–156 Table 5 The EcLeuRS mutant that is specific for (2S)-3-[(6-acetylnaphthalen-1-yl)amino]-2-aminopropanoic acid (32); aaRS, aminoacyl-tRNA synthetase. aaRS Residue

38

40

41

499

500

527

537

538

541

560

Substrate

Reference

EcLeuRS Anap-2C

Leu Phe

Met Gly

Leu Pro

Tyr Val

Tyr Leu

Tyr Ala

His Glu

Leu Ser

Phe Cys

Ala Val

Leu 32

UniProtKB P07813 (SYL_ECOLI) Lee et al. (2009)

to analyze the effects of side-chain packing on protein folding and stability. Again, Brustad et al. chose the same active site residues as Wu et al. (2004) (see above) to generate their EcLeuRS mutant library. They introduced the mutations Thr247Val/Thr248Val or Thr252Phe into the editing domain of EcLeuRS to improve the ncAA incorporation efficiency and/or fidelity. These mutations did indeed result in more model protein labeled with 9 but they provoked a slightly higher background incorporation of cAAs in the absence of the analog as well. This is in contrast to the finding of Lemke et al. (2007) who observed an increase in fidelity when they introduced the Thr252Ala mutation into the editing domain of EcLeuRS (see above). In addition, Brustad et al. included an o-pair expression construct with three copies of the EctRNALeu5 CUA in their analysis. However, the increase in suppressor tRNA copy number did not improve the incorporation efficiency (Brustad et al., 2008). Ai et al. expanded the palette of ncAAs with long aliphatic side chains to analogs with alkenyl functions. Amino acids with alkene moieties are particularly interesting for bioorthogonal conjugation reactions, as for instance olefin metathesis (see the references in Ai et al. (2010) and reviewed by Kim et al. (2013)). Ai and coworkers started from the o-pair Cap2X/EctRNALeu5 CUA that had been developed for long-chain ncAAs by Brustad et al. (2008). Being an EcLeuRS descendant, Cap2X contained mutations in the active site residues as suggested by Wu et al. (2004) (see above). Another Glu20Lys mutation was also present that had not been included in the original library (Brustad et al., 2008). Ai et al. additionally randomized residues Thr247, Thr248, and Thr252 of the editing domain. Applying the established positive/negative selection (Chin et al., 2003b) on their new library, they selected the o-pair AK-1/EctRNALeu5 CUA (mutations are shown in Table 4) that was promiscuous for (2S)2-aminohept-6-enoic acid (21), (2S)-2-aminooct-7-enoic acid (22), L-O-crotylserine (28), and L-O-(pent-4-en-1-yl)serine (29). Using this new o-pair, they assessed the incorporation of compounds 21, 22, 28, and 29 into a GFP(Y39am) mutant (Wang and Wang, 2008) in an NMD-deficient upf1D knockout strain. The background incorporation of cAAs in the absence of an ncAA was low and no contamination with cAA was observed when the analogs were added to the medium at a concentration of 2 mM. They obtained between 3 mg/L and 6 mg/L of the different variants (see Table 2 for details). To show the applicability of alkene amino acids for bioorthogonal conjugation, they incorporated 28 with the new o-pair at two amber mutations in circularly permutated yellow fluorescent protein (cpVenus(R168am L178am)). Residues Arg168 and Leu178 were chosen as the insertion sites of 28 because they were spatially close to each other. The vicinity of the two residues allowed the formation of an olefin bridge between the adjacent alkene side chains by an intramolecular metathesis reaction (Ai et al., 2010). In the future, this technique could be employed to produce stable and protease-resistant peptides. Summerer et al. applied the established methods for the generation of EcLeuRS libraries (Wu et al., 2004) and positive/negative selection (Chin et al., 2003b) to generate an o-pair that was specific for the fluorescent amino acid (2S)-2-amino-3-({[5-(dimethyla mino)naphthalen-1-yl]sulfonyl}amino)propanoic acid (31) (Summerer et al., 2006). Again, the Thr252Ala mutation improved the fidelity of the o-pair. In accordance with Chen et al. (2007b) Summerer and coworkers removed the 30 -CCA from EctRNALeu5 CUA (EctRNA0 Leu5 CUA ) and equipped the truncated tRNA gene with the

50 - and 30 -flanking sequences of the SUP4 gene (SUP4-EctRNA0 Leu5 CUA ). As proposed by Chen et al. (2007b) three tandem copies of the 0 Leu5 SUP4-EctRNA0 Leu5 CUA chimera ([SUP4-EctRNA CUA ]3) were expressed under the control of the PGK1 promoter for an improved expression of the suppressor tRNA (see Section 3.1). The LeuRSB8T252A/[SUP4-EctRNA0 Leu5 CUA ]3 pair (mutations are shown in Table 4) was used to incorporate 31 at two different positions of hSOD. 31 served as an environmentally sensitive reporter of local structural transitions during unfolding. When the hSOD(W33 [31])-His6 (see Table 2 for titer) and hSOD(Q16[31])-His6 mutants were denatured the change of fluorescence intensity and emission wavelength of 31 reflected the structural rearrangements at these positions (Summerer et al., 2006). Lee et al. explored another structural probe for S. cerevisiae proteins (Lee et al., 2009). (2S)-3-[(6-acetylnaphthalen-1-yl)amin o]-2-aminopropanoic acid (32) is an ncAA that carries a highly environmentally sensitive fluorophore in the side chain. Lee et al. incorporated it into the glutamine-binding protein (QBP) of E. coli using the Anap-2C/EctRNALeu5 CUA o-pair that was derived from the EcLeuRS/EctRNALeu5 CUA pair. To evolve the specificity for 32 it was necessary to extend the mutagenesis of the EcLeuRS beyond the usual five residues (Wu et al., 2004) to other amino acids in the active site (see Table 5; refer to Table 2 for the titer of hSOD(W33[32])His6). 32 was incorporated in response to an amber mutation at position Asn160 of QBP. Asn160 is sensitive to conformational changes that are induced when L-glutamine binds. QBP Asn160 [32] was expressed in NMD-deficient upf1D S. cerevisiae. Binding of glutamine to the isolated variant blue-shifted the fluorescence of 32 and its intensity increased, however, the fluorescence anisotropy before and after glutamine binding did not significantly change. Lee and coworkers interpreted the changes in fluorescence by differences in the polarity of the environment around 32 when glutamine was bound or not (Lee et al., 2009). Metal ions are involved in a number of biological processes and ncAAs could provide new chemistries for the design and engineering of redox-active centers containing metal ions. Tippmann et al. first incorporated a Fe-ncAA into a protein in S. cerevisiae (Tippmann and Schultz, 2007). Following the strategies outlined above, they evolved two o-pairs for L-S-ferrocenylcysteine (27) Leu5 starting from the EcLeuRS/EctRNALeu5 CUA pair. LeuRS32/EctRNACUA and LeuRS35/EctRNALeu5 (see Table 4 for mutations) were able to CUA accommodate the large hydrophobic side chain of 27. The Fecontaining ncAA was efficiently incorporated into hSOD (W33am)-His6 and 0.25–0.5 mg/L of hSOD(W33[27])-His6 was produced (Table 2). Tippmann and Schultz demonstrated successful incorporation of a metallo-ncAA and it is likely that other metal-carrying ncAAs can be introduced into proteins as well. In the future, proteins containing metallo-ncAAs could be applied in electron transfer processes, e.g. from electrode surfaces to catalytic sites (Tippmann and Schultz, 2007). 3.2.3. O-pairs for S. cerevisiae that are derived from pyrrolysyl-tRNA synthetase PylRS and its corresponding suppressor tRNAPyl CUA A naturally occurring orthogonal PylRS/tRNAPyl CUA pair directs the incorporation of the lysine analog pyrrolysine (33) in response to an in-frame amber stop codon in Methanosarcina species and a few bacterial species (Gaston et al., 2011). Pyl The MmPylRS/MmtRNAPyl CUA and MbPylRS/MbtRNACUA pairs are

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Table 6 MbPylRS mutants specific for Pyl/Lys analogs. Residues in bolditalic were not included in the mutagenesis; aaRS, aminoacyl-tRNA synthetase.

a b

aaRS Residue

241

266

267

270

271

274

313

Substrate

Reference

MbPylRS AcKRS a TfaKRS b PCKRS (clone 39)

Met Met Met Phe

Leu Met Leu Leu

Ala Ala Ala Ser

Leu Ile Ile Leu

Tyr Phe Leu Cys

Leu Ala Ala Met

Cys Phe Phe Cys

Pyl 35 36 37

GenBank AKB58742.1 Neumann et al. (2009) Neumann et al. (2008) Gautier et al. (2010)

Identical to AcKRS-3 described by Neumann et al. (2009); contains the additional mutation D76G, which was not programmed. Identical to AcKRS-2 described by Neumann et al. (2008).

orthogonal in E. coli and have been successfully used to incorporate a palette of structurally and chemically diverse ncAAs into proteins in E. coli (reviewed by Wan et al. (2014)). Mukai and coworkers introduced the MmPylRS/MmtRNAPyl CUA into yeast, however, the performance of the pair was weak (see the supplementary information of Mukai et al. (2008)). The group of Chin made a systematic attempt to add 33 and its analogs to the genetic code of S. cerevisiae. They first replaced the EcTyrRS/EctRNATyr CUA pair in their expression vector (Fig. 2A) by the MbPylRS/MbtRNAPyl CUA pair and assayed its activity with N6-[(propargyloxy)carbonyl]-L-lysine (34) in the reporter strain MaV203 (Fig. 1). 34 is a known substrate for wildtype MbPylRS (Wan et al., 2014). While the MbPylRS was expressed the MbtRNAPyl CUA was not transcribed, most probably due to the lack of consensus A- and B-boxes (Hancock et al., 2010). Hancock et al. tried all strategies that had previously been applied Leu5 to improve the transcription of EctRNATyr CUA and EctRNACUA . They Pyl mutated the sequence of the MbtRNACUA to introduce a nearconsensus A- and B-box each. This improved the transcription of the suppressor tRNA only incrementally and only the combination of the A- and B-box mutations resulted in low transcription levels. The production of MbtRNAPyl CUA could be improved by fusion to the SNR52 promoter containing extragenic A- and B-boxes (see Section 3.2.1) but still did not support robust growth of the reporter strain MaV203 on -His medium with the standard 3-AT concentration (Chin et al., 2003b) in the presence of 34. Next, they fused the 0 MbtRNAPyl CUA downstream to the 5 -flanking region of yeast tRNA genes that harbor an extra upstream TATA-box promoter element for enhanced transcription when the A- and B-boxes are separated by large intervening sequences such as introns. However, to no avail, MaV203 did not show the phenotypes of ncAA-active and cAA-inactive o-pairs in the presence and absence of 34 (Fig. 1). Only Asp when Hancock et al. exchanged MbtDNAPyl CUA for tDNAGUC of the Arg Asp bicistronic tDNAUCU-tDNAGUC gene substantial amounts of MbtRNAPyl CUA were transcribed. The transcription of this bicistronic gene is controlled entirely by the A- and B-boxes of the upstream gene tDNAArg UCU. The two genes are transcribed as one precursor from which the mature tRNAs are released by posttranscriptional processing. However, the mature MbtRNAPyl CUA produced in this way was not orthogonal in S. cerevisiae. Hancock and co-workers mutated G3U70 to A3-U70 and thus converted the MbtRNAPyl CUA to MmtRNAPyl CUA. This finally brought about the desired orthogonality Pyl of the suppressor tRNA. The SctDNAArg UCU-MmtDNACUA construct was co-expressed with different MbPylRS mutants that had been previously evolved in E. coli (Gautier et al., 2010; Neumann et al., 2008) (see Table 6) to incorporate L-N6-acetyllysine (35; a mimic of posttranslational modification) and its fluorinated analog 36 as well as the photocaged Lys derivative 37 into hSOD(W33am)Pyl His6. The MbPylRS/SctDNAArg UCU-MmtDNACUA pair facilitated the incorporation of the photocrosslinker amino acid 38 (Chou et al., 2011). hSOD(W33am)-His6 variants labeled with 34, 35 and 36 were isolated in tens of lg per liter culture (Table 2). 3.2.4. Site-specific incorporation of ncAAs in other yeasts than S. cerevisiae Young et al. introduced the site-specific labeling of proteins with ncAAs into P. pastoris (Young et al., 2009). Next to its adoption

in mammalian cells for cell biology studies, the implementation of the technique in P. pastoris represents a major advancement since the methylotrophic yeast is an industrially important eukaryotic production host for recombinant proteins. In P. pastoris, Young et al. (2009) exploited the EcTyrRS mutants that had been successfully evolved and applied in S. cerevisiae for the incorporation of para-iodo-L-phenylalanine (11), para-acetyl-L-phenylalanine (13), para-benzoyl-L-phenylalanine (14), and O-methyl-L-tyrosine (16) ((Chin et al., 2003a), see Table 3) as well as of para-azido-Lphenylalanine (12) ((Chin et al., 2003a; Deiters et al., 2003), Table 3) and para-propargyloxy-L-phenylalanine (17) ((Deiters et al., 2003), Table 3). In addition, EcLeuRS mutants with a specificity for the photocaged serine analog 30 ((Lemke et al., 2007), Table 4) and for the fluorescent ncAA 31 ((Summerer et al., 2006), Table 4) were functional in P. pastoris. Young et al. assayed several P. pastoris promoters for the expression of the ncAARSs and found that the FLD1 promoter was optimal for an efficient amber suppression. To further improve the expression of the ncAARS they added an enhanced Kozak sequence (Kozak, 1999) to the 50 -end of the coding region (Fig. 2D). For the expression of the suppressor tRNAs, they used the chimeric [SUP4-EctRNA0 Tyr CUA]3 and [SUP4-EctRNA0 Leu5 CUA ]3 constructs under control of the PGK1 promoter that Chen et al. (2007b) had successfully developed for S. cerevisiae (see Section 3.2.1; Fig. 2D). The o-pairs were integrated into the genome of P. pastoris. For the functional analysis of the o-pairs in P. pastoris, Young and co-workers employed an amber mutant of the recombinant human serum albumin (rHSA(E37am)). The 24 amino acid ‘‘prepro” leader sequence of the model protein was compatible with the P. pastoris protein secretion system and facilitated the export of the labeled variant proteins into the medium. They attained substantially higher protein titers than those previously reported for S. cerevisiae. rHSA(E37am) labeled with the ncAAs mentioned above accumulated to 10–40% of the wildtype rHSA (see Table 2 for details). rHSA(E37[13]) was successfully decorated with the antiangiogenic peptide ABT-510 by a bioorthogonal oxime ligation between the unique keto-group of 13 and a complementary reactive alkoxyamine moiety on ABT-510 (Young et al., 2009). The group of Sohn added the reactive ncAA para-azido-Lphenylalanine (12) to the genetic code of the pathogenic fungus Candida albicans (Palzer et al., 2013). They demonstrated the utility of the ncAA for light-induced protein interaction mapping in C. albicans at the examples of the highly abundant oxidative stress response protein Tsa1p and the less abundant transcription factor Tup1p. The azido-group of 12 was intact and available for photocrosslinking after incorporation into target proteins in C. albicans as confirmed by Staudinger ligation (Köhn and Breinbauer, 2004) of Tsa1p(N141[12]) with a triarylphosphine-functionalized fluorescent dye in cell extracts (Palzer et al., 2013). Palzer and coworkers used the p-azidoPheRS-1 evolved by Chin et al. (2003a) (Table 3) for the incorporation of 12 in S. cerevisiae. However, C. albicans uses an alternative genetic code and decodes CTG codons as serine instead of leucine (Santos and Tuite, 1995). Accordingly, functional expression of p-azidoPheRS-1, which is a descendant of the TyrRS from E. coli, only occurred after codon optimization of the gene for C. albicans (Palzer et al., 2013). The

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authors also adapted the expression of the suppressor tRNA for C. albicans. Drawing on the observations of Wang and Wang (2008) in S. cerevisiae, (Palzer et al., 2013) removed the 30 -CCA from EctRNATyr CUA and fused it to the SNR52 promoter and the terminator of glutamic acid tRNA (tECUC) of C. albicans. Four copies of the SNR52-EctRNA0 Tyr CUA-tECUC cassette were fused in tandem and integrated into the C. albicans genome at the SNR52 locus. The codon optimized p-azidoPheRS-1 was integrated at the ACT1 locus under control of the strong ACT1 regulating sequences (Palzer et al., 2013). The site-specific incorporation of ncAAs in C. albicans could be particularly useful to study host-pathogen interactions.

4. Conclusion More than 30 ncAAs (Table 1) have been incorporated successfully into proteins in different yeasts such as S. cerevisiae, P. pastoris, and C. albicans. Their structures and chemistries are diverse, many of them are analogs of the aromatic amino acids Phe, Trp, and Tyr (1, 2, 10–17). As well, derivatives of the aliphatic amino acids Ala (18–22), Met (3–9), Cys (23–27), Ser (28–30), and Lys (34–38) were added to the genetic code of yeasts. The photocrosslinker ncAAs 14 and to a lesser extent 12 were extensively used to study protein–protein interactions in S. cerevisiae (Berg et al., 2014; Chen et al., 2007a; Huang et al., 2008; Mohibullah and Hahn, 2008; Shiota et al., 2011; Tamura et al., 2009; Yamano et al., 2010) and C. albicans (Palzer et al., 2013). The analogs 12, 13, 17, and 28 provided reactive handles to bioorthogonally conjugate proteins from yeast to fluorescent dyes (Deiters et al., 2003), to polyethylene glycol (Deiters et al., 2004), and to functional peptides (Young et al., 2009), or to install protein side chain bridges (Ai et al., 2010). The fluorescent analogs 31 and 32 were demonstrated as environmentally sensitive structural probes (Lee et al., 2009; Summerer et al., 2006). The photcaged Cys and Ser analogs 26 and 30 were useful to photocontrol enzyme activity (Wu et al., 2004) and subcellular protein trafficking (Lemke et al., 2007) in yeast. The redox-sensitive metallo-ncAA 27 could be used to engineer yeast proteins with designed redox centers in the future (Tippmann and Schultz, 2007). The Se-derivative of Met, 4 finds application in the crystallography of yeast proteins to solve the phase problem (Bushnell et al., 2001; Malkowski et al., 2007). Many different target proteins can be labeled with ncAAs in yeast. The W33 amber mutant of human superoxide dismutase, hSOD(W33am)-His6, has been the preferred model protein for the evolution of new orthogonal pairs in yeast (Ai et al., 2010; Brustad et al., 2008; Chen et al., 2007b; Chin et al., 2003a; Deiters et al., 2003; Hancock et al., 2010; Lee et al., 2009; Lemke et al., 2007; Nehring et al., 2012; Summerer et al., 2006; Tippmann and Schultz, 2007; Wu et al., 2004). The Q16am and N27am mutants of hSOD were employed to study positional effects of ncAA incorporation (Chen et al., 2007b). GFP (Wang and Wang, 2008) and other fluorescence proteins such as cpVenus (Ai et al., 2010) were also used as model proteins. The engineering of proteins with ncAAs in yeast was extended to enzymes such as CalB (Budisa et al., 2010), RNA polymerase II (Chen et al., 2007a) and caspase 3 (Wu et al., 2004), as well as to membrane proteins, for instance the GPCR Ste2p (Huang et al., 2008) and to the proteins of the mitochondrial translocator complex (Shiota et al., 2011; Tamura et al., 2009; Yamano et al., 2010). Furthermore, different ncAAs were introduced into recombinant human serum albumin (Young et al., 2009), the co-chaperone Aha1 (Berg et al., 2014), the oxidative stress response protein Tsa1p (Palzer et al., 2013), the transcription factor Tup1p (Palzer et al., 2013), and into glutamine-binding protein (QBP) (Lee et al., 2009). Protein titers range from tens of lg/L (0.05 mg/L hSOD(W33[13])-His6 (SCS),

153

(Chin et al., 2003a); 0.05 mg/L hSOD[7] (SPI), (Wiltschi et al., 2008)) to more than 150 mg/L (rHSA(E37[13]), (Young et al., 2009)). While the residue-specific incorporation of ncAAs in E. coli has become a routine (Johnson et al., 2010) and is also quite common for proteome profiling in mammalian cells (Hinz et al., 2013; Ngo and Tirrell, 2011), the technique is still rather exceptional in yeasts. It finds its most important application in yeast protein crystallography by the substitution of Met with 4. Malkowski et al. (2007) and Kitajima et al. (2010) successfully developed remedies for the toxic effects of 4 (Section 2.1), which improved the labeling efficiency of yeast proteins with this analog. The residue-specific incorporation of Met analogs (Wiltschi et al., 2008) and fluorinated aromatic amino acids (Budisa et al., 2010) was demonstrated. However, the protein titers as well as the incorporation efficiencies need further improvement, for instance by the co-expression of aaRS and tRNAs or by the thorough control of the incorporation processes in the bioreactor (Section 2.1). The site-specific incorporation of ncAAs has found broad application in yeast. A powerful selection system in S. cerevisiae facilitates the evolution of new o-pairs for virtually any ncAA and the expression systems of the latest generation allow for their efficient production (Chen et al., 2007b; Wang and Wang, 2008). Current o-pairs for yeasts are descendants of the orthogonal Leu5 EcTyrRS/EctRNATyr CUA and EcLeuRS/EctRNACUA pairs from E. coli or the archaeal MmPylRS/MmtRNAPyl pair and MbPylRS. Although CUA the transcription of yeast tRNAs differs from those of E. coli or archaea, sophisticated strategies have been developed for the transcription of functional orthogonal tRNAs. These involve the fusion of the tRNA coding sequences, for instance, to the 50 - and 30 -flanking sequences of the yeast SUP4 gene (Chen et al., 2007b); or to RNA promoters with upstream A- and B- boxes such as of SNR52 (Wang and Wang, 2008); or their integration into chimeric Pyl bicistronic tRNA genes as in the SctDNAArg UCU-MmtDNACUA construct (Hancock et al., 2010). With these tools at hand, ncAAs with their extraordinary chemistries have become a useful asset in the protein engineering toolkit for yeasts. Besides the extraordinary traits they can introduce into recombinant proteins, they promise exciting new opportunities to study cellular processes in yeast. Acknowledgments This work has been supported by the Federal Ministry of Science, Research and Economy (BMWFW), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, the Government of Lower Austria and ZIT – Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG (Grant number 282482). Support by CHEM21 in the frame of the Innovative Medicines Initiative Joint Undertaking under grant agreement no 115360, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007–2013) and EFPIA companies’ in-kind contribution is acknowledged. References Ai, H.-W., Shen, W., Brustad, E., Schultz, P.G., 2010. Genetically encoded alkenes in yeast. Angew. Chem. Int. Ed. Engl. 49, 935–937. Allison, D.S., Han Goh, S., Hall, B.D., 1983. The promoter sequence of a yeast tRNAtyr gene. Cell 34, 655–663. Anderson, J.C., Wu, N., Santoro, S.W., Lakshman, V., King, D.S., Schultz, P.G., 2004. An expanded genetic code with a functional quadruplet codon. Proc. Natl. Acad. Sci. USA 101, 7566–7571. Annaluru, N., Muller, H., Mitchell, L.A., Ramalingam, S., Stracquadanio, G., Richardson, S.M., Dymond, J.S., Kuang, Z., Scheifele, L.Z., Cooper, E.M., Cai, Y.,

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